Automated Enzyme Assays

Automated enzyme assays

LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY

Edited by T. S . WORK - N.I.M.R., Mill Hill E. WORK - Imperial College London

Advisory board G. POPJAK - U. C. L. A . S. BERGSTROM - Stockholm K. BLOCH - Hurvard University P. SIEKEVITZ - Rockefeller University E. SMITH - U. C. L. A . E. C. SLATER - Amsterdam

NORTH-HOLLAND PUBLISHING COMPANY -AMSTERDAM * LONDON

AUTOMATED ENZYME ASSAYS

D. B. Roodyn Department of Biochemistry University College London

1970 NORTH-HOLLAND PUBLISHING COMPANY -AMSTERDAM

*

LONDON

0 1970 North-Holland Publishing Company All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanicaI,photocopying, recording or otherwise, without the prior permission of the copyright owner Library of Congress CataIog Card Number: 68-54514 ISBN North-Holland: 0 7204 4206 0

Published by: NORTH-HOLLAND PUBLISHING COMPANY - AMSTERDAM

Sole distributors for the U.S.A.and Canada: AMERICAN ELSEVIER PUBLISHING COMPANY, INC.

52 VANDERBILT AVENUE, NEW YORK, N.Y. 10017

This book is the pocket-edition of Volume 2, Part I, of the series ‘Laboratory Techniques in Biochemistry and Molecular Biology’. Volume 2 of the series contains the following parts: Part I Automated enzyme assays by D. B. Roodyn Part n Cellulosic ion exchangers by E. A. Peterson

Printed in The Netherlands

Contents

Preface .

............................. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter I .Principles and terminology of enzyme automation . . . . Chapter 2 . Semi-automatic methods . . . . . . . . . . . . . . .

26

.............. ..............

26 29

2.1, Detection of enzyme-catalyzed reactions 2.2. Examples of semi-automaticmethods .

7 9 13

Chapter 3. Automatic methods illustrated by the Techniconsystem

.

35

3.1. The Technicon autoanalyzer . . . . . . . . . . . . . . . . . . 3.1.1. Proportioning pump 3.1.2. Sampler 3.1.3. Manifold . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.5. Recording devices . . . . . . . . . . . . . . . . . . . . . . 3.1.6. Dialyzer . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.7. Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.8. Heating bath . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Automation of a simple-enzymeassay . . . . . . . . . . . . . . 3.2.1. The manual method . . . . . . . . . . . . . . . . . . . . . 3.2.2. Dilution factors and fractional volumes 3.2.3. Adaptation to the autoanalyzer . . . . . . . . . . . . . . . 3.3. The sampling process 3.4. Interference by protein 3.4.1. Continuous dialysis 3.4.2. Continuous filtration . . . . . . . . . . . . . . . . . . . . .

.

35 36 37 37 37 38 38 38 39 39

..................... ..........................

.

............. . ...................... ..................... .....................

40 41

42 50 54 54 55

.

AUTOMATED ENZYME ASSAYS

4

.

Chapter 4 Interrupted-Jow and discrete-samplingsystems

....

..................... ....................

4.1. Interrupted-flow systems 4.2. Discrete-sampling systems

.

................ 5.1. Molar conversion factors and enzyme units . . . . . . . . . . . . Chapter 5 Single-enzyme analysis

................. ................ ..... .......................

5.2. Determination of incubation time 5.3. Measurement of the progress curve 5.4. Relation between enzyme activity and protein concentration 5.5. Enzyme monitoring

.

59 59 63

68 68 71 75 80 81

..........

87

.......... ........................ .....................

87 92 96 98

...............

102

Chapter 6 Multiple-enzyme analysis (M.E.A.) 6.1. Principles of multiple-enzyme analysis (M.E.A.). 6.2. Enzyme patterns . . . . . . . . . . . . . . . 6.3. Enzyme gradients 6.4. Multi-channel analyzers

Chapter 7. Enzyme characterization

.........

. . . . . . . . . . . . 102 106 ..... 107 ........................ 108 ...................... . . . . . . . 108

7.1. Substrate specificity and Michaelis constants 7.2. p H optimum . . . . . . . . . . . . . . . . . . . . . 7.3. Enzyme stability 7.4. Temperature optimum 7.5. Plots of multi-parameter studies . . . . . . . . . .

Chapter 8. Calculation of enzyme activities from instrument 114 readings . . . . . . . . . . . . . . . . . . . . . . . 114 ........................ . . . . . . . . . . . . . . . 118 . . . . . . . . . . . . . . 119 122 ....................... . . . . . . . . . . . . . . . . . . . 124 . . . . . . . . . . 125 . . . . . . . . 126 131 .................... . . . . . . . . . . . . . 131

8.1. Data processing 8.2. Calibration and calculation of results 8.2.1. Molar conversion-factor calculation 8.2.2. Incubation time 8.2.3. Line-volume calculations 8.2.4. Calculation of concentrations of line reagents 8.2.5. Gradient calibration and gradient-making systems 8.2.6. Protein concentration 8.2.7. Sample pattern and theory of sampling 8.2.8. Calculation of enzyme activity

.................

138

5

CONTENTS

......

140

9.1. Flow system . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Properties of the system . . . . . . . . . . . . . . . . . . . . . 9.2.1. Incubation time . . . . . . . . . . . . . . . . . . . . . . 9.2.2. Fractional-line volumes 9.2.3. Sample interaction and wash characteristics . . . . . . . . . . 9.2.4. Gradient-making system . . . . . . . . . . . . . . . . . . 9.2.5. Preparation of reagents . . . . . . . . . . . . . . . . . . . 9.3. Computer program . . . . . . . . . . . . . . . . . . . . . . . 9.3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2. Molar conversion-factor calculation 9.3.3. Incubation time and chart-speed determinations 9.3.4. Line-volume calculations . . . . . . . . . . . . . . . . . . 9.3.5. Line-reagent and reaction-mixture calculations . . . . . . . . . 9.3.6. Stock solutions . . . . . . . . . . . . . . . . . . . . . . 9.3.7. Gradient calibration 9.3.8. Assay conditions . . . . . . . . . . . . . . . . . . . . . . 9.3.9. Protein concentration 9.3.10. Sample pattern . . . . . . . . . . . . . . . . . . . . . . 9.3.11. Results 9.3.12. Computer sub-routines . . . . . . . . . . . . . . . . . . . 9.4. Example of the use of the above system . . . . . . . . . . . . . .

140 143 143 144 144 145 148 149 149 149 150 150 150 150 150 151 151 151 152 152 152

Chapter 9. Generalized systemfor enzyme automation

...................

............. ........

.................... ................... ..........................

Conclusions and future prospects

. . . . . . . . . . . . . . . . . 165

. . . . . . . . . 168 Appendix II . Terminology used in enzyme automation . . . . . . . 173 Appendix III . Apparatus used in enzyme automation . . . . . . . 176

Appendix I . Published automated enzyme assay#

111.1. Analytical systems and instruments

................

111.1.1. Automated analytical systems . . . . . . . . . . . . . . . . 111.1.2. Spectrophotometers. colorimeters and their accessories 111.1.3. Other automatic sensing devices . . . . . . . . . . . . . . . 111.1.4. Dispensers. diluters, pumps and sampling systems . . . . . . . 111.1.5. Reagents 111.2. Addresses of firms

.....

......................... .......................

176 177 182 187 189 190 191

6

AUTOMATED ENZYME ASSAYS

.

Appendix ZV Computer program for generalized enzyme automation system . . . . . . . . . . . . . . . . . . . 193

. . . . . . . . . . . . . . . . . . . 193 . . . . . . . . . . . . . . . . . . . 195 . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

IV.1. Input: general instructions Iv.2. Input: detailed instructions IV.3. Program

............................ Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . .

References

208 218

Preface

This book is primarily intended to be of some help to those who wish to free themselves of the burden of repetitive manual enzyme assays. Because of the current rapid developments in the field of biochemical automation, it is not intended as a comprehensive account of all the available systems, but rather as a guide to present trends. The literature in the subject is scattered over a wide range of journals, technical reports, brochures, reports of meetings and many other publications that are not usually found in department libraries. I must therefore apologize for any serious deficiencies in the text or for incompleteness in the material presented in the appendices on methods and instrumentation. I have illustrated the text heavily from experiments drawn from my own experience with the Technicon autoanalyzer, which has inevitably placed considerable weight on continuous flow methods. However, I have tried to overcome this bias by treating the subject in as general a manner as possible, emphasizing the principles of the various analytical approaches, rather than the detailed procedures. In such a young subject there is still no generally accepted terminology, and from necessity I have developed my own, which I include in an appendix. There is still much to be done in elaborating a theoretical basis for the subject of automated enzyme assay, and I have made a hesitant start in this direction in ch. 8. I have included a generalized system of analysis, with a full computer program, at the end of the

I

Sublecl index P. 218

8

AUTOMATED ENZYME ASSAYS

book in the hope that the approaches I have evolved in my own work may be of some use to others. As I have intimated in the last chapter, it is possible that the rapid development of automated techniques in enzymology will have a revolutionary effect on the subject of Biochemistry as a whole and on related disciplines, and may well drastically change the working patterns of scientists in these fields. Combined with the inexorable developments in computer technology, these advances may well result in systems of biochemical analysis that are quite foreign to our present generation of biochemists. Perhaps this book may be of some help in the runaway world in which we may soon find ourselves.

November 1969

D. B. Roodyn

Introduction

Although the study of enzyme catalyzed reactions has been a central part of Biochemistry since its earliest days, it is only recently that methods have been developed which can largely eliminate the tedium of repetitive enzyme assays. At the present time there is considerable interest in automation of enzyme assay and many alternative systems have appeared on the market recently. Although the design of these systems differs considerably, the principles involved in their use are common to all. The aim of this book is to discuss these principles so that whatever the actual instrument used, the reader will be able to develop an automated assay with maximum ease and precision. A list of published automated assays is given in appendix I. At the moment, the majority of such assays have been performed with the Technicon autoanalyzer. For convenience, therefore, I have illustrated the text with various experiments from our laboratory using the Technicon system. However, the results and methods have been presented in a general manner applicable to any automated system. Once the principles of enzyme automation have been acquired, the reader may well find it more convenient to develop his own automated assay, rather than to search the literature for published methods to adapt to his own specific problems. A generalized method for automated enzyme assay is presented in detail in ch. 9 which applies to many current manual assays. The range of instruments now available for automated enzyme 9

Subfect Jndex P. 218

10

AUTOMATED ENZYME ASSAYS

assays is shown by the material in appendix 111. There are several reviews on the subject (Bodansky and Schwartz 1961; Stein et al. 1965; Schwartz and Bodansky 1966,1968; Wacker and Coombs 1969). There is a wealth of information in the various reports of the Technicon symposia (‘Automation in Analytical Chemistry’, Mediad Press, New York, 1965-1967) and the Technicon Corporation has also published two useful bibliographies (‘Bibliography of Automated Enzyme Research’ 1965; ‘Technicon Autoanalyzer Bibliography 1957/ 1967’). The use of various biochemical analyzers has recently been reviewed by Buckley (1969). Reference to these sources and the detailed references in appendix I will help the reader in automating manual assays. It is probable that the introduction of fully-automated enzyme assay systems into the general field of enzyme research will have a dramatic effect. It is now possible to perform in a day experiments that would have previously taken weeks. This may be illustrated by an example of one of our experiments.* Suspensions of cells of four different mutants of yeast were disrupted with glass beads in a high speed shaker. The homogenates so obtained were then assayed in a dual-channel multipleenzyme analyzer. Four enzymes were estimated, each at 4 different protein concentrations and for 2 different times of incubation. Each assay had an appropriate blank assay. The results were taken from the recorders and fed into a digital computer which determined in each case the enzyme activity in pmoles/ml, pmoles/ml/min and pmoles/mg dry wt./min. In these calculations the computer used information on instrument calibrations and molar extinction coefficients that was obtained before the assay. The total number of assays, including blanks, was 256 and about 30 instrument calibration values were used. Since the computer corrected for instrument zeros, and blanks, and interpolated molar extinction coefficient values, the number of computational steps was several thousand. All the results were tabulated and printed in graphical form, with suitably calculated scale divisions.

* Experiments reported from our laboratory were supported by grants from the Science Research Council.

INTRODUCTION

11

All these operations were performed in about 5 hr, and during much of the time the operator was free to do other tasks. A reasonable estimate for the time required for a similar ‘manual’ experiment would have been about a week to 10 days. The full potentialities of such systems are perhaps not yet appreciated. Clearly, where routine and standard assays are performed on large numbers of enzyme samples (as in clinical laboratories) automation with computer processing of data must result in a great improvement in the operational efficiency of the laboratory. The use of enzyme assays on tissue fluids as a diagnostic aid is now well established (Quastel 1961) and it is in the clinical field that automated analytical methods have had their greatest impact so far (Whitby et al. 1967). There is little doubt that the introduction of these methods in clinical laboratories has been most beneficial. For example, Robinson et al. (1 966) have compared manual, ‘work-simplified’, and autoanalyzer techniques by work-study methods. They conclude that where there is a large number of assays to perform, the autoanalyzer is greatly superior in accuracy and reproducibility. This was found to be mainly due to the effect of tedium on the human operator during repetitive assays. (Of course it does not follow that automation is always better. For example, Nothstein and Ellerbrook (1962) used the autoanalyzer reagents for the assay of serum alkaline phosphatase to develop a rapid manual method that they regarded as more convenient than the automated procedure!) We can say therefore, that certainly in clinical laboratories, automated methods have in general proved themselves. The impact of fully-automated analytical systems on pure research laboratories has not yet been fully felt. Since such laboratories have their share of tedium no doubt the benefits will be as great. I must clarify a bias in this book. It is addressed mainly to those who wish to assay enzymes, rather than to enzymologists. I hope I will not cause any offence by saying that not all biochemists who estimate enzymes are enzymologists. Going further one might venture to suggest that not all those who estimate enzymes are even biochemists. Although this state of affairs may be disturbing to some purists, it is not at all suprising, or even unwelcome. Enzymes are major compoSubject index P. 218

12

AUTOMATED ENZYME ASSAYS

nents of living matter. Their activity, distribution and interaction are subject to the most complex influences and are fundamental to the behaviour of all cells. Used as ‘markers’, they can monitor the sedimentation of sub-cellular particles with great precision. In clinical assays, changes in the enzyme activity of tissue fluids may be the first indication of pathological change. Their levels in micro-organisms may determine and act as indicators of the nature of the fermentation products. Changes in their electrophoretic pattern may reveal genetic abnormalities. Study of their activity during cell differentiation and morphogenesis may reveal fundamental processes in cell growth. Thus, the determination of enzyme activities in living systems is a fundamental and universal weapon of all biologists. Industrial enzymology is now expanding rapidly and attracting wide interest, including soappowder manufacturers. If we define enzymology as the study of the structure and function of enzymes with a view to explaining the latter in terms of the former, we must conclude that it is responsible for only a part of the total volume of work now being carried out on enzymes. For this reason, this book is designed for the non-specialist whose main aim is to determine the level of enzyme activity in suitable preparations of biological materials. For a general treatment of enzymes, the reader is recommended to read ‘Enzymes’ by Dixon and Webb (1964). Other useful books are ‘An Introduction to the Study of Enzymes’ by Gutfreund (1965) and ‘Structure and Function of Enzymes’ by Bernhard (1968). There are also several detailed textbooks on enzyme assay in general. These include ‘Methods of Enymatic Analysis’ edited by Bergmeyer (1968) and the series ‘Methods in Enzymology’ edited by Colowick and Kaplan (1955 onwards). A useful source of references on automated enzyme assays is ‘Analytical Abstracts’, published by the Society for Analytical Chemistry.

CHAPTER 1

Principles and terminology of enzyme automation

Apart from enzymes which have characteristic adsorption spectra (such as the cytochromes), the overwhelming majority of enzymes are estimated by means of their catalytic activity. Provided that the enzyme is saturated with substrate, the rate of reaction is directly proportional to enzyme concentration (see Bodanksy 1959) for a useful introduction to enzyme kinetics in relation to problems of enzyme assay). The rate of reaction is the rate of disappearance of substrate or appearance of product. A plot of concentration of substrate or product against time is called a progress curue and the rate is the slope of this curve. For a variety of reasons (including depletion of substrate, possible product inhibition, thermal inactivation or change in pH) the progress curve is seldom linear over its entire course. It is therefore usual practice to measure the initial slope of the progress curve. Thus, valid enzyme assays require that the enzyme be fully saturated with substrate and that the initial rate is measured. It is not always possible to work under zero order conditions,i.e., under conditions in which the substrate concentration does not affect the rate of reaction. For example, cytochrome oxidase and catalase assays are based on a first order reaction, with the rate of reaction falling continuously as the substrate is depleted.In these situations, the enzyme activity must be expressed in terms of the first order velocity constant. Whilst it is desirable to perform assays under theoretically acceptable conditions, it may not always be essential. If the aim of the experiment is to determine the absolute enzyme activity of a sample in terms of 13

Sublccr index P. 218

14

AUTOMATED ENZYME ASSAYS

pmoles/min, then these criteria have to be fulfilled. However, there are many situations in which the point of interest is not so much the absolute activity, as the change in relative activity. For example, if one is following the inactivation of an enzyme with time, the results may be expressed adequately as percent of initial value. During purification of an enzyme, the values of interest are the relative specific activities of samples and starting material. If one is studying levels of enzymes in a bacterial culture under different growth conditions, again percentage changes are of greater interest than absolute values. In these situations, the prime requirement is that the reading obtained shouldbeproportiona1 to enzyme concentration. Whilst one can assure this by working with a fully-saturated enzyme and measuring initial rates, it may be possible to obtain proportionality with respect to enzyme concentration without fulfilling either of these criteria. An example is given in fig. 5.9. where one can obtain proportionality with non-linear progress curves. As regards substrate concentration, Bodansky (1953) developed a method whereby meaningful measurements could be made at sub-optimal substrate concentrations. The autoanalyzer was first calibrated with a standard reference enzyme, and then all unknown enzymes were estimated by determining the amount of reference enzyme which would produce the same change in substrate concentration in the same time. Pitot et al. (1965) discuss the effect of order of reaction on automated assays and assess the various criteria that should be met in such assays. One may conclude, therefore, that although it is preferable to use saturating amounts of substrate and to measure true initial rates, it may be possible in certain other circumstances to make useful measurements. This applies particularly to continuousflow systems, in which measurements of true initial rate may be difficult (see 0 5.2). In general all enzyme assays consist of the following operations : 1. The reagents are prepared from solids or stock solutions. These include buffer, substrate, and any co-factors required for the reaction to proceed. 2. Reagents are mixed in suitable proportions to give the reaction mixture.

PRINCIPLES AND TERMINOLOGY

15

3. The enzyme is mixed with the reaction mixture and incubated in some suitable incubation vessel. 4. The progress of the reaction is followed either by the disappearance of substrate or the appearance of product(s). The reaction is followed by some suitable sensing device or sensor*, which can detect and measure the physical or chemical changes that occur during the reaction. 5. The response of the sensor is recorded in some way so as to provide a permanent record of the reaction. If the sensor contains a device for making this record (e.g. an amplifier connected to a pen recorder) it may be called an autosensor*. 6 . The reaction is stopped. 7. Calculations are performed on the record so as to convert instrument readings into suitable units, such as pmoles/min. The sequence of events is shown in fig. 1.1, and the extent to which the events are automated in various systems is shown in fig. 1.2. The order of events may be changed for various reasons. For example it is usual practice to add substrate to reaction mixture and start the reaction by addition of enzyme. This is because many enzymes are unstable if incubated in the absence of substrate. However, if the enzyme preparation is a crude tissue extract it may well contain significant amounts of ‘endogenous’ substrates. In such cases, more meaningful assays are obtained by short pre-incubation in the absence of substrate. Each assay has its own particular difficulties. For example, if the enzyme preparation is a suspension of particles (e.g. mitochondria) it may be necessary to prevent aggregation or clumping of the particles during the assay. Thus the stirring must be sufficient to maintain a homogeneous reaction mixture, but not too violent to cause damage to the particles. The problem of blanks with crude enzyme preparations, such as serum, can often be serious and appropriate allowance has to be made for such factors as the presence of endogenous substrates, instability of substrates under the conditions of incubation (e.g. the See appendix I1 for terminology used in this book. Subject index p . 218

16

AUTOMATED ENZYME ASSAYS

Stock solul ons and solids -~~ Calculate dilution of stock or required weight of solid. Prepare solutions ~

1

Reagents reagents and enzyme to give required assay conditions. Mix reagents in Reaction mixture e.g., time of incubation, temperature

I

Incubation vessel ~

I

Select suitable settings on sensor, e.g., wavelength, slit width

Sensor Adjust recording device, e.g., baseline and chart speed of recorder

Record Calculate enzyme activity in suitable units from values on record, e.g., from

I

Final result From result decide conditions required for next assay

Next experiment

Fig. 1.1. General sequence of events in enzyme assays. Main events areindicated by arrows, and detailed operations are given in rectangles. Fig. 1.2. Methods of automating sequence of events given in fig. 1.1. Main events are as in fig. 1.1. Methods of automating the various operations are given in rectangles.

+

PRINCIPLES A N D TERMINOLQGY

17

Stock solutions and solids Weighing by automatic balance. Dilutions by autodiluters or proportioning pumps. Calculations by computer c

Reagents Dilutions by autodiluter or proportioning pumps. Mixing by jets or mixing coils. Addition of samples by samplers. Calculations by computer

Selection of settings done by operator. In ‘closed loop’ system could be done by on-line computer. Transfer to sensor by probes or pumps

,

-

Variety of recording devices available to monitor output of sensor. Adjustments done by operator but could be done by on-line computer

Fig. 1.2.

I

Subject index R. 218

18

AUTOMATED ENZYME ASSAYS

spontaneous hydrolysis of labile esters), the release of interfering substances from the enzyme preparation itself (e.g. the release of bound inorganic phosphate from the enzyme during a phosphatase assay) and, of course, drift in the recording or sensing device (e.g. drift in the base-line, or slow deposition of protein on optical surfaces). One of the great advantages of automated methods of enzyme assay is that it is very easy to make large numbers of measurements of these blanks at various stages of the assay with little addition to the workload, a situation that is not the case in manual assays. It should be noted that enzyme assays are essentially repetitive operations. Thus after the operations 1-7 above have been completed, they are repeated with modification in one of the operations. For example, if the aim is to determine the enzyme activity in a number of different samples, such as serum samples in a clinical laboratory, the cycle would be repeated with new enzyme samples added at stage 3. If several substrates are used for each enzyme preparation, as in multiple-enzyme analysis (see below), an ‘inner’ cycle would be used so that each enzyme preparation would be tested against a cycle of reaction mixtures. We may now consider different types of assay in more detail. Assays may be divided into two primary classes: ‘single-enzyme analysis’ (S.E.A.) and ‘multiple-enzyme analysis’ (M.E.A.), depending on whether one or several enzymes are being assayed. Multiple assay methods are relatively recent and are still in the process of active development (see ch. 6 ) and the bulk of the literature on enzyme assays is orientated towards the assay of individual enzymes. S.E.A. may be used for a variety of purposes, as follows. It often happens that a newly discovered enzyme, or a known enzyme in a new tissue, needs to be ‘characterized’. This usually entails measurement of Michaelis constant (I&,), p H optimum, temperature optimum, stability at 0 “C and during the assay, substrate specificity, response to inhibitors etc. Such studies proceed by using the same enzyme and varying the composition of the reaction mixture, the concentration and type of substrate, and the conditions of incubation. They are usually intended to provide what are called ‘optimum’ conditions of assay and are primarily performed for reasons of convenience, as a prelude to further studies

PRINCIPLES AND TERMINOLOGY

19

on the enzyme. More detailed and extensive studies of this nature amount to pure enzymological research, and have, of course, given us valuable information about the mechanism of enzyme action. However, as mentioned in the introduction, treatment of such detailed work is outside the scope of this book. Some systems for the automation of the simpler stages in the characterization of enzymes are given in ch. 7. Another obvious use for S.E.A. is in enzyme purification. This needs little discussion. Steps 1-7 are repeated using samples of enzyme at different stages of the purification process. This type of work introduces the question of determination of protein concentration during enzyme assays. An estimate of protein concentration is of the greatest importance in interpretation of enzyme assays. In enzyme purification, the value needed is the specific activity, which is the enzyme activity per unit of protein. There has been a great deal of confusion about enzyme units, and the International Union of Biochemistry suggested in 1961 that the units should be pmoles per minute per mg protein (’Enzyme Nomenclature’, 1965). To quote the 1965 report: ‘One unit (U) of any enzyme is that amount which will catalyse the transformation of 1 micromole of substrate per minute ... under standard conditions.’ Also: ‘The concentration of an enzyme in solution (as distinct from its purity) will normally be given as units per ml.’ ‘The specijic activity of an enzyme preparation is defined as units per mg protein. It is directly related to the purity of the preparation .’ Another measurement that may be made is pmoles of substrate transformed per ml of reaction mixture in a given time (i.e. other than 1 min). This value, which I will call ‘total reaction’, is useful in automated systems in which the precise time of incubation may be difficult to determine. Its use sometimes has advantages. For example, suppose we are following a reaction by measuring reduction of mM NAD’ at 340 nm. The total reaction will give a measure of percent reduction of NAD’; thus a value of 0.9 pmoles/ml will correspond to 90% reduction. If the total reaction value is divided by the incubation time, this relationship is obscured. If the incubation time were 2 min, the Subject index P. 218

20

AUTOMATED ENZYME ASSAYS

enzyme concentration (in pmoles/ml/min) would be 0.45 units. The degree of purification of an enzyme is assessed from specific activity values, and the amount of enzyme, or its recovery during purification from concentration and total reaction values. In work on crude enzyme preparations, such as suspensions of subcellular particles, the measurement of both specific activity and total reaction or enzyme concentration is essential to assess the recovery of particles, the cross-contamination between particles, and the general validity of the fractionation scheme. (An analysis of the types of enzyme assay performed in sub-cellular fractionation is given in Roodyn (1965a).) It is generally essential to have some parameter to which we relate enzyme measurements. This is usually total protein, although in some situations, for example in rapidly dividing cells in which the overall composition of the cell changes during growth, enzyme activity per unit DNA may be more meaningful. In other situations, enzyme activity per unit dry weight, or per unit volume of culture, or per cell may be preferable. Even the measurement of specific activities on a protein basis may have its hazards, because of the difficulty of obtaining an accurate estimate of protein. Thurman and Boulter (1966) discuss various methods for estimating proteins and it is clear that no singlemethod is completely satisfactory.Methods based on total nitrogen give results that depend on the amino acid constitution and amide content of the protein. Colorimetric methods, such as the Folin-Ciocalteau or biuret methods, give different colour yields with different proteins and are subject to interference by a variety of agents. The measurement at 280 nm is notoriously unspecific. Dry weight measurements are affected by the degree of hydration which is not easy to control. In fact, it may not be too extreme to say that the only really meaningful measure is the total amino acid composition of the protein! At the moment, measurement of enzyme activity and protein are carried out as separate operations, often at different times. However, automated systems for the simultaneous measurement of both protein and activity have been described (e.g. Scheuerbrandt 1965) and their more general use would be of great benefit.

PRINCIPLES AND TERMINOLOGY

21

Perhaps the most common use for automated S.E.A. is what may be called enzyme sample determination. Most of such work is at present performed in clinical laboratories, where methods particularly suited to the tissue fluids under test have been evolved. Although pure and applied research laboratories often need to estimate numbers of enzyme samples, there is generally not the steady and routine flow of material to be tested that there is in clinical laboratories. Again, in the research laboratory, it is more usual to alter the conditions of assay from one experiment to another, and detailed inspection of the progress curve is more frequently required. For these reasons, the tendency in such situations has been to develop ‘semi-automatic’ systems (see ch. 2) in which there is a considerable degree of human intervention in the assays. However, it may well be that the development of flexible and generalized enzyme automation systems (see ch. 9) may facilitate the process of ‘enzyme sampling’ in general. Indeed it may not be unusual in the future for several laboratories to share a central ‘enzyme assay centre’, analogous to a computer centre, in which samples brought with a particular assay program are analysed automatically by a specialist staff not under the direct control of the user. The possible implications of central analytical laboratories is discussed later in ‘Conclusions and future prospects’. All the applications given above in S.E.A. can also be performed on groups of enzymes by multiple-enzyme analysis, M.E.A. This approach is relevant when the sample contains several enzymes, and it is suprising how frequently this occurs. Tissue fluids, homogenates, suspensions of sub-cellular particles, impure enzyme preparations, microbial cultures, may all be subjected to such analysis. Being a multiple analysis, particular problems arise in the assay procedure which must often strike a balance between practicality and precision. It is often difficult to arrange assay conditions which are optimum for every enzyme in the group, and sometimes it may be necessary to work under sub-optimum conditions in order to gain the advantages of a group assay. The subject is discussed in detail in ch. 6 ; I will merely stress at this point that the ability to estimate groups of enzymes automatically adds another dimension to the techniques of Subiecf index p . 218

22

AUTOMATED ENZYME ASSAYS

enzyme assay and opens up the possibility of performing extremely complex analyses of biological materials. I would like now to try to define more precisely what I mean by an ‘automated’ enzyme assay. Strictly speaking, a fully automated assay is one in which dl1 the processes (1-7, pp. 14-15, and fig. 1. l ) are performed automatically. If the analyzer is connected to a digital output device and hence to a computer, the operator merely introduces reagents and enzyme into the machine and in due course receives a print-out of the enzyme activity in suitable units. As such systems are by no means universal, 1 would call them ‘fully-automated assays’, to distinguish them from the more common ‘automated assays’, in which data processing is not performed ‘on-line’. An automated assay system must prepare the reaction mixture from suitable stock solutions, add the substrate, equilibrate at the required temperature if necessary, add the enzyme, mix it with reaction mixture, carry out the incubation, with stirring if required, follow the course of the reaction, either by directly measuring some property of the reaction mixture, or by stopping the reaction, removing the protein and examining the filtrate or dialysate, and, finally, record the reaction in some permanent form (e.g. on a pen recorder or paper tape). The task of the operator is to prepare the reagents and enzyme, start and stop the analysis, and perform the required calculations on the final record (this may then be done ‘off-he’by an automated data processing system). A ‘semiautomated assay’ is one in which only some of the above operations are performed automatically. For example, if the reaction mixture, substrate and enzyme are pipetted manually into the cuvette of a recording spectrophotometer, the assay may be regarded as semiautomated, even though the progress curve is followed automatically (in one, or several cuvettes). There have recently been developed a large number of devices of varying complexity for dilution and sampling of reagents; methods based on such apparatus are sometimes called ‘work-simplified’. The combined use of such methods with semi-automated assays blurs the distinction between semi- and full automation. At the moment, however, the distinction is fairly clear and provides a useful basis for treatment of the subject. Probably in

PRINCIPLES AND TERMINOLOGY

23

the near future laboratories will have equipment which allows a continuous progression from manual to automated. The ‘manual assay’ is reasonably easy to define as one in which none of the operations 1-7 are performed automatically. For example the reaction mixture and enzyme are pipetted into a test-tube, shaken on a water bath, deproteinized, centrifuged, and the supernatant removed and read in a manual colorimeter. However, again the division between ‘manual’ and ‘automatic’ pipetting has become blurred recently. For example, Harrison (1966) describes a semi-automatic pipette to deliver 106) pl and 200 pl samples with & 1% accuracy. The BTL Dilutronic 250 (Baird and Tatlock Ltd., Chadwell Heath, Essex) contains two recycling syringes: one takes up sample into the sample pipette, and the other takes diluent into the diluent pipette; sample and diluent are then expelled into the receiving vessel, with a predetermined dilution (see appendix 111 for further details). Schwartz and Bodansky (1963) talk of three stages in automation. In stage I the reaction mixture is prepared manually and the measurement of enzyme activity is recorded in an instrument. In stage 11, the reaction mixture is prepared automatically. Stage 111 ‘... would incorporate feedback devices for controlling and correcting the action of the instrumental components and transformation of the enzyme activities into numerical values’. These correspond roughly to my three classes of ‘semi-automated’, ‘automated’ and ‘fully automated’. However, stage 111 differs in the important respect that it introduces the concept of feed-back control. This raises the possibility of a level of automation far above even the fully automated systems. There is much current discussion of ‘closing the loop’, i.e., developing a system in which an on-line computer adjusts the operation of the instrument on the basis of the output from the instrument and not by command from the operator. At a simple level, the analyzer could decide whether to repeat an assay or alter the assay conditions (perhaps by increasing the enzyme concentration if tl e rate is too low). However, at a higher level, the system could perform multiple assays, arrange its own work-load, data-processing and filing systems, and control its operation not only on the basis of the immediate result, but also by access to Subiecr index P. 218

24

AUTOMATED ENZYME ASSAYS

a central store of analytical data. For example, the general request may be: ‘Of the enzyme assays available, find out which enzyme in this sample is most active and determine its characteristics.’ The system would rapidly scan the assays built into it, determine the most active enzyme, refer to its information store as to the properties of that enzyme, prepare appropriate reaction mixtures and carry out a flexible analysis of the enzyme based on its response to each condition of assay. Because of the very high ‘data-yield’ of fully-automated systems, it may become increasingly difficult for the human operator to assess and collate the output of the analytical system and he will inevitably have to rely to a greater extent on the computer in this task. Thus, what at the moment appears speculative, may well become a reality because of the problems of processing large amounts of analytical data. Another division of enzyme assays is into ‘continuous-flow’ and ‘interrupted-flow’methods. In the former method, all the operations proceed sequentially and without interruption. These systems, at first sight a little strange to the biochemist, are in fact interesting analogues of steady-state systems in the living cell. In the interruptedflow systems, some of the operations, i.e., preparation of the reaction mixture and introduction of the enzyme are carried out sequentially. The flow system is then stopped and the reaction is monitored with a stationary reaction mixture in the reaction vessel, which is usually the cuvette of a spectrophotometer. Such systems have great advantages for following progress curves and hence determining true initial rates. A third system has recently been widely introduced. This is the so-called ‘discrete-sampling’ analyzer. Each assay is performed in its own reaction vessel (usually a disposable plastic tube) with reagents introduced at various time intervals by suitably positioned automatic pipettes. These analyzers generally are able to operate with very little inter-sample contamination. In general it is difficult to monitor the progress curve continuously with continuous-flow analyzers or discrete-sampling systems, although there have been various arrangements to do this, at least in a somewhat primitive fashion (e.g. by the measurement of a small number of

PRINCIPLES AND TERMINOLOGY

25

different incubation times with different sensors). It is surprisingly difficult to obtain detailed information about the entire progress curve with available continuous-flow systems. This is because the enzyme and substrate are reacting in a continuously flowing stream. The only way to follow the course of the reaction is to set up ‘monitoring points’ along the stream. A complete sensing device is required at each monitoring point, since the same device cannot be used at the same time at more than one place. This would make the cost of obtaining, for example, six readings along the progress curve quite considerable. Thus at present, cofitinuous monitoring is best done in interruptedflow systems.

Subjecf index p . 218

CHAPTER 2

Semi-automatic methods

2. I . Detection of enzyme-catalyzed reactions In most semi-automatic methods the reaction mixture is prepared manually. A suitable volume is placed in the reaction vessel, the enzyme is added manually, mixed with the contents of the reaction vessel and then the sensor is allowed to follow the course of the reaction automatically (auto-sensor). A large number of methods have been used to follow enzyme-catalyzed reactions since almost any chemical or physical change that can be measured in a recording instrument may be used. For full details of the methods used the reader is referred to the textbooks mentioned at the end of the introduction. Gutfreund (1965) gives a useful account of the various approaches, and some of his examples will be considered. The following brief account is not intended as a comprehensive survey, but merely as a demonstration of the wealth of methods available. The most usual method is to measure changes in light absorption and a variety of instruments have been used for this purpose, from simple filter colorimeters t o sophisticated spectrophotometers. Changes in visible or ultra-violet light occur either as a direct result of the reaction or as a consequence of chemical or physical treatments carried out on the reaction mixture. Perhaps the commonest measurements are at 650-700 nm for the blue complex formed after the reduction of phosphomolybdate in the assay of inorganic phosphate, and at 340 nm to follow the reduction of NAD+ or NADP'. Many reactions have been linked either directly or indirectly to such measure26

SEMI-AUTOMATIC METHODS

27

ments. Esterases are often measured by using substrates with chromogenic substituents (e.g. nitrophenyl groups) which give characteristic colours after release from the substrate, as in the use of p-nitrophenyl phosphate in the assay for phosphatases. There are many systems available for monitoring changes in light absorption, and a commonly used instrument is the Gilford 2000 multiple sample absorbance recorder (appendix 111, p. 183). Wood and Gilford (1961a) give full details of such a system. If the enzyme has a characteristic absorption spectrum itself, as with haem proteins, it is possible to assay the enzyme, and even to follow the formation and disappearance of the enzyme-substrate complex by direct spectrophotometric measurement. A semi-automated system based on spectrophotometric measurement is described by Henry et a]. (1963) for use in clinical enzymology. Another important method of studying enzyme-catalyzed reactions is by measuring changes in fluorescence. In his excellent book on ‘Fluorescence Assay in Biology and Medicine’, Udenfriend (1962) discusses the use of such measurements in enzymology and demonstrates their widespread application and great sensitivity. Fluorimetry is also used to measure common substrates and co-factors, by the use of specific enzymes. As with light absorption methods, there is a great range in sophistication of the instruments used. Fluorescence spectroscopy is extremely sensitive and is useful, for example, in estimating reduced pyridine nucleotides. Duysens and Amesz (1957) used it to follow levels of NADH in living cells. For rapid reaction studies, stopped-flow fluorimetry is of use (see, for example, Harvey 1969, and Chen et al. 1969). A variety of electrode systems have been used, including electrodes sensitive to changes in concentration of dissolved oxygen, COz, Na+, or K + ions and, of course, H + ions. As an example, Severinghaus and Bradley (1958) described a double electrode for measuring pOz and p C 0 2 in biological fluids. The oxygen electrode is widely used in studies on mitochondria and oxidative enzymes. In p H measurements, the actual change in p H may be followed; in a more convenient method the p H is maintained constant by the continual addition of acid or alkali, and the volume of titrating fluid plotted against time gives a Subject index P. 218

28

AUTOMATED ENZYME ASSAYS

measure of the rate of the reaction (see for example the rapid pH-stat assays described by Dyson and Noltmann (1965). Gutfreund and Hammond (1963) in a discussion on the use of pH assays to follow enzyme activity stress the sensitivity and simplicity of such methods. Other physical methods have been used. For example, changes in the heat content of the reaction mixture may be exploited, as in the calorimetric measurements of biological oxidations by Estabrook and Gutfreund (1960). Magnetic measurements may be used, for example in a study of myoglobin by Brill et al. (1960). There have been many studies on enzymes using NMR (nuclear magnetic resonance); Shulman (1962) summarized the use of electron and nuclear spin resonance in biology and gave some examples of its application to enzymology (see also Beinert and Palmer 1965). A very sensitive method for enzyme assay that is being used increasingly is to measure the conversion of a radioactive substrate to a radioactive product (for a detailed account see Reed 1968). The radioactive product is separated from the substrate by a variety of means, and if the separation is efficient (i.e. there is no contamination with residual substrate which would invalidate the assay), the sensitivity is quite remarkable. For example, with a substrate of specific radioactivity 62 mC/milliatom, one can detect 3 x 10- l 2 moles of product! The method has certain difficulties however. Apart from the problem of achieving a clear separation between substrate and product, the specific activity of the substrate must be known precisely, and the substrate must be extremely pure. (I am not aware of any fully-automated methods based on radioassay. However, if developed, their potential should be very great.) There is now a considerable literature on the separation of enzymes by various forms of electrophoresis, for example on gels or cellulose acetate strips. These studies are of particular importance in the study of isoenzymes. Usually the enzymes are detected qualitatively by treatment with a reaction mixture or dye that gives a coloured or UV-absorbing product, but quantitative methods may be used. As an example, Opher et al. (1966) studied the isoenzymes of serum lactate dehydrogenase by strip electrophoresis. They incubated the strips

SEMI-AUTOMATIC METHODS

29

with substrate and tetra-nitro-blue tetrazolium. This was reduced to a purple formazan by the action of the enzyme, and the amount of reduction was proportional to enzyme concentration. Quantitative determination of the coloured area was carried out with a densitometer; the assay may therefore be regarded as a semi-automated procedure.

2.2. Examples of semi-automatic methods These, then, are some of the methods available for the study of enzyme activity. How are they used as semi-automatic methods? In the simplest device, a temperature-controlled cuvette containing reaction mixture is placed in a recording spectrophotometer. A ‘base-line’ is obtained for any absorption by the reagents and then substrate and enzyme are added. For example a suitable volume of buffer is placed in the cuvette, NAD’ and malate are then added, followed by malate dehydrogenase. A trace of increase in absorbance at 340 nm with time is obtained (fig. 2.1) and the malate dehydrogenase activity may be

Chart divisions (inch)

Fig. 2.1. Example of automatic recording of progress of an enzyme catalyzed reaction. Semi-automatic assays for malate dehydrogenase with a recording spectrophotometer. The reaction mixture contained malate and NADf at pH 9.9. At arrows, successive samples obtained during chromatographic purification of the enzyme were added to a succession of cuvettes manually. The vertical lines were caused by closing the shutter between the addition of samples. (See Roodyn et al. (1 962) for details.) Subject index I. 218

30

AUTOMATED ENZYME ASSAYS

calculated from the initial slope of the curve. This type of system is very useful if one wishes to make various additions while the reaction is in progress, with the amount and type of substance added depending on the initially observed rate. It is used a great deal, for example, in studies on mitochondria1 metabolism with the oxygen electrode. Quite complex traces may be obtained in this way. Provided that the reaction does not proceed too rapidly, the research worker can alter the composition of the reaction mixture at will by addition of small volumes of reagent while observing the progress of the reaction on the chart. For example, he can increase the amount of a possible inhibitor until the required inhibition is reached. On the basis of the instrument response, he can add any of his other reagents in any sequence at any time. This type of ‘man-machine’ interaction can be very fruitful. In more fully automated systems the sequence of operations has to be predetermined to a far greater extent, with the machine operation having to be clearly programmed before the assay begins. It would be useful to be able to alter the operation of the machine according to the results being obtained (see ‘closing the loop’, p. 23). However, the controls of almost all analytical systems at the moment are still under operator rather than computer control. Patient (1960) discussed the general problem of the relative advantages of partial and complete automation by taking examples from the processes involved in the manufacture of penicillin. It was found that total automation was expensive and required extensive instrumentation and control processes. However, if only those parts of the system that required a high degree of precision were automated and other activities (such as the loading of tube-racks) given to unskilled operators, a less expensive and more flexible system was possible. Thus in all automated systems a balance must be struck between the ideal and the practical. The introduction of systems for recording the reaction in a single incubation vessel was clearly an advance on manual methods. However, even in such simple systems, it does not follow that the manual approach is always inferior. Unless one has as many sensors as samples to be assayed, it could well be quicker to prepare a rack of test-tubes manually and incubate them all simultaneously, rather than to carry

SEMI-AUTOMATIC METHODS

31

out the assays sequentially with a recording device. For example, let us suppose that a given enzyme requires 3 min to give a convenient reading. If 100 samples are to be analysed, the time for sequential analysis is at least 300 min, not allowing for the time taken to load each sample into the recording device. If it takes one minute to pipette the sample and reaction mixture into a test-tube, about 100 min may be spent preparing racks of tubes manually. These could all be incubated simultaneously (perhaps for 5 min). If the tubes could be read manually at a rate of one every 30 Eec, the entire manual assay could take about half the timc of the semi-automatic assay. If the rate of reaction is slow, and it takes 10-20 min to obtain a satisfactory reading, the difference in time becomes even greater. To overcome this difficulty, manufacturers of recording spectrophotometers have introduced automatic sample changers of various degrees of complexity. Usually, about 4 cuvettes are loaded into the machine on a movable carriage, and each cuvette is moved in front of the light path at intervals determined by some attached control device. An early model for an automatic cuvette positioning attachment for a spectrophotometer is described by Wood and Gilford (1961b). It is usually difficult, or impossible, to add reagents to the cuvettes of the automatic sample changer, while the reaction is in progress. (Some instruments with this facility have become available recently : see appendix 111.) These systems are therefore useful when the assay procedure is reasonably standard and there is no need t o add reagents as the reaction progresses. Some automatic sample changers operate with flow cells, but usually the cuvettes are filled manually. Generally it is difficult to stir the reaction mixture in the cuvettes, although recently Beckmann-Spinco Instruments have described a system which does this (Kintrac VII: see appendix 111, p. 182). An automatic stirring device for four cuvettes has also been described by Lysionok and Flynn (1969). Since the cuvette itself is the reaction vessel, certain limitations are imposed on the type of reaction that can be followed spectrophotometrically. For example, continuous aeration of the liquid would be impossible because of light scattering from the bubbles. There may also be problems with temperature control and Subject index P. 218

32

AUTOMATED ENZYME ASSAYS

with turbidity of the enzyme if it is bound to cell particles. The AmincoChance dual-wavelength spectrophotometer is designed to measure optical density changes in very turbid solutions (appendix 111,p. 184). It is theoretically preferable to carry out the enzyme reaction in a separate vessel and to pump a small fraction of the reaction mixture continuously through a continuous flow autosensor. The reaction vessel could then be designed specifically for the type of enzyme reaction followed and limitations imposed by the geometry of the cell carriage would be overcome. It should be noted that automation of the sensing device does not completely remove the tedium of repetitive assays, and as we have seen, in some cases may have certain disavantages. Nevertheless such instruments represent an important half-way stage between manual techniques and full automation by eliminating human error from the actual measurement of the enzyme-catalyzedprogress curve. It is thus becoming increasingly rare to see biochemists rapidly jotting readings as they simultaneously follow a stop-watch and the needle of the spectrophotometer, Several recording manometer systems have been described, usually based on the well-known Warburg manometer. For example Raikhman (1966) described a system in which the level of fluid in the manometer is detected by a suitable electrode system (see appendix 111, p. 188, for other examples). In many ways such instruments have advantages over recording spectrophotometers. A larger number of samples can be assayed at the same time. Temperature control is rigorous and it is possible to agitate and gas the vessels during the course of the reaction. It is interesting how fashion may sometimes dictate the equipment purchased. Many ingenious and fruitful techniques of manometry were developed before 1939 to follow enzyme reactions, for example using bicarbonate buffers in a COz gas phase to monitor acid-base changes (Dixon 1951). These methods are now generally neglected in favour of the recording spectrophotometer. Perhaps a more widespread use of recording manometers, or devices such as the Gilson respirometer (see appendix 111, p. 188) will correct this tendency. The oxygen electrode attached to a suitable circuit and recorder is a

SEMI-AUTOMATIC METHODS

33

semi-automatic device that has had a great impact on our knowledge of the biochemistry of respiratory systems. The reaction mixture is fully aerated and then the vessel is usually sealed from the air after addition of enzyme, so that as the reaction proceeds the oxygen concentration in the reaction mixture falls. This means that after a time, measurements are being made a t low oxygen tensions, and by the end of the experiment the vessel becomes virtually anaerobic. Thus experiments performed immediately after sealing the reaction vessel are not necessarily identical to apparently similar experiments performed later. This again illustrates the difficulty that may arise from having to use the sensor as the reaction vessel. Pressman (1967) employed a multiple incubation vessel into which are inserted an oxygen electrode, and electrodes for measuring H + , Na+ and K+ ions. At the same time the turbidity of the preparation can be measured. The plot includes a trace in which the rate of oxygen uptake is obtained by a simple electronic circuit that differentiates the signal from the oxygen electrode. With the availability of multichannel recorders and X-Y plotters, further development of such ‘multi-parameter’ sensor systems will be facilitated. By providing simultaneous measurement of different properties of the reaction mixture, more meaningful data may be obtained than from sequential measurements. For certain enzymes, particularly the flavoproteins and cytochromes, direct measurement of the spectrum is extremely useful. There have been many studies on the spectral changes that occur during the catalytic cycle of such enzymes, and these changes have been most useful in the study of the formation of enzyme-substrate complexes (for example see the high-resolution spectrophotometric studies of Chance (195 1) with catalase). Lundegardh (1959) described the spectrophotometric techniques available at that time for the study of respiratory enzymes in the living cell, and since then more complex systems have been developed. The study of enzyme-substrate complexes also calls for extremely rapid measurements. These are often performed in a stopped-flow apparatus which can measure reactions that occur in the millisecond range. For example, Gibson and Milnes (1964) give the Subject index p . 218

34

AUTOMATED ENZYME ASSAYS

detailed design of a stopped-flow apparatus for following reactions with half-times of 5 msec upwards. The main features in the design of the Durrum stopped-flow apparatus are given in appendix 111, p. 184. One of the problems of studying enzyme-catalyzed reactions is that if the protein concentration is too high the rate may become too high to measure. Thus most studies are made at low enzyme concentration and high substrate concentration. However, in the cell, the reverse situation may be nearer reality, and it is important to understand the processes that occur at high enzyme concentrations. Eigen and Hammes (1963) in a general review on the study of fast reactions in enzymology describe the use of relaxation methods. In these the effect of a sudden perturbation to the system is examined (Eigen 1968), and it is possible to perform such studies at the desired high enzyme concentration. In a general sense, one could regard the monitoring of a reaction mixture with an autoanalyzer as a semi-automatic method, with the analyzer acting as a chemical sensor. For example Lenard et al. (1965) followed the progress of amidase- and peptidase-catalyzed reactions by pumping the reaction mixture continuously into an autoanalyzer that carried out the ninhydrin reaction. At the moment such systems are not widely used. However, if the reaction mixture could be monitored at the same time by other sensors (e.g. various electrodes) most useful analytical methods would result. For example, simultaneous monitoring of oxygen concentration and levels of inorganic phosphate in mitochondria1 suspensions would provide a continuous measure of P/O ratios. Thus the combined use of multi-xensor reaction vessels with chemical analyzers could greatly increase the productivity of semi-automatic methods.

CHAPTER 3

Automatic methods illustrated by the Technicon system

3.1. The Technicon autoanalyzer In compiling appendix I, the author has found that, a t the moment, the majority of published methods for enzyme automation are based on the Technicon system. It is therefore convenient to introduce the principles of automated assay by reference to this system. Several continuous flow systems have been described in the literature. For example, Jonnard (1960) described a ‘Random Selection System for Dynamic Biochemical Analysis’ which consists of a fluid processing system, mixer, sampler, colorimeter, various programmers, an automatic titrator, amplifier and recorders. However, the most commonly used continuous system is the Technicon autoanalyzer based on the method of Skeggs (1957) in which the earliest designs of sampler, proportioning pump, dialyzer, heating bath and colorimeter are given. The Technicon system has been used for enzyme assays for at least 10 years (e.g. Marsh et al. 1959) and appendix I demonstrates the range of enzymes that have been automated with this method. As with much current equipment, the Technicon system is based on the use of ‘modules’. Instead of purchasing a single complex instrument that is specifically designed for the required purpose, one can purchase sub-systems which may be assembled in various combinations to suit individual needs. Also, other equipment may be combined with one or more of the modules. The Technicon modules that are generally used for enzyme assays are as follows: 35

Subjecr index P. 218

36

AUTOMATED ENZYME ASSAYS

3.1.1. Proportioning pump A series of plastic tubes are clamped against a flat surface by means of two end blocks and are compressed by rotating metal bars. The volume of fluid pumped by each tube, or line is determined by the internal diameter of the pump tubing. If the pump tube is L cm in length and has a diameter of d cm, the flow rate for a bar moving along the tube at b cycles/min is given by

LV =lrd2bL/4,

(3.1)

where LV is flow rate or line volume in ml/min. Since many different TABLE3. I Pumping rates with Technicon tubing. (Values refer to clear standard tubing used with a standard-speed proportioning pump. Pumping rates should not fall below the minimum value or rise above the maximum value. The expected rate is given by the nominal value, and above 0.32 ml/min there is usually little deviation from this value.) Colour code

Orange black Orange red Orange blue Orange green Orange yellow Orange white Black Orange White (clear) Red Grey Yellow Yellow blue Green Purple Purple black Purple orange Purple white

Pumping rate (ml/min) Minimum

Nominal

Maximum

0.005

0.015 0.030 0.050 0.10 0.16 0.23 0.32 0.42 0.60 0.80 1 .oo 1.20 1.40 3.00 2.50 2.90 3.40 3.90

0.029 0.048 0.072 0.128 0.19 0.27 0.36 0.47 0.66 0.87 1.08 1.28 1.49 2.10 2.63 3.03 3.54

0.016 0.032 0.075 0.13 0.19 0.28 0.37 0.54

0.73 0.92 1.12

1.31 1.90 2.37 2.77 3.26 3.15

4.05

THE TECHNICON SYSTEM

31

diameters of tubing are available, a wide range of volumes may be delivered at one pumping speed. The available range for the ‘standard’ proportioning pump is given in table 3.1. With a variable speed pump, however, any pumping speed may be obtained (see 0 9.2.2). 3.1.2. Sampler A circular table carries up to 40 cups and a probe dips into the cups at pre-determined time intervals. In the early sampler module (sampler I) the probe can sample at 20, 40 or 60 cups/hr, and between samples the probe aspirates air for half the sampling time. Thus at 60 cups/hr the probe is in the sample for 40 sec and in air for 20 sec. The improved sampler (sampler 11) has a wider range of sampling times and has a system for washing the probe between samples. It can also be refrigerated so that it is preferable for enzyme sampling. A larger sampler is available, but is not generally used for enzyme assays. 3.1.3. Manifold This is a system of tubes, glass joints, mixing coils and other devices that is attached to the pump, receives the various reagents and the samples, mixes them in various proportions determined by the line volumes in the proportioning pump and then delivers the mixtures to various peripheral devices. The manifold also usually contains waste tubes returning from these devices to the input (or ‘venous’ side) of the pump and hence to the waste receiver or drain. The manifold is usually mounted on a plastic tray fitting over the pump and can be detached more or less intact, so that the same analyzer may be used for different assays. The term manifold is sometimes also used in the more general sense to describe the entire flow diagram of the analytical system. 3.1.4. Sensors A variety of continuous-flow sensors are manufactured by Technicon specifically, but provided that a de-bubbling device is fitted, most other continuous-flow sensors may be used. The most commonly used device is the Technicon double-beam colorimeter which selects wavelengths by suitable filters. This is generally adequate for most Subierr index P. 218

38

AUTOMATED ENZYME ASSAYS

assays in which some coloured product is formed and may also be used for measurement of NADH and NADPH, although this is rather at the optical limits of the lamp. For UV measurements, or more precise spectral measurements, a spectrophotometer is required. The other device of increasing use in enzymology is the continuous-flow fluorimeter, and Technicon have recently introduced an improved version with greater stability and a very small flow cell. Alternatively various electrodes or flame photometers may be used. The factors of importance in selecting a continuous-flow autosensor for automation are the sensitivity, the retention volume of the flow cell and the stability. 3.1.5. Recording devices These are usually supplied with the sensor, although flexible arrangements are possible. For example, White and Gauger (1968) have used the Gilford Model 300 spectrophotometer with a Technicon autoanalyzer and ratio recorder. The commonest recording device is the pen-recorder ; Technicon supply one-, two- or three-pen recorders. It is becoming increasingly common to have some form of digital output (e.g. paper tape), either in addition to, or instead of, the pen recorder. These systems are discussed in detail in § 8.1.

3.1.6. Didlyzer This dialyzes the stream from the manifold against an appropriate medium and is used to separate low-molecular weight substances (such as phosphate) from protein for subsequent assay. The efficiency of concurrent dialysis is generally not very high, so that it cannot be used to separate all the low-molecular weight materials from the protein. (Much more efficient dialysis can be achieved by ‘counter current’ dialysis in which the donor and acceptor streams flow in opposite directions. However, in this case it would not be possible to perform assays on material crossing the dialysis membrane.) 3. I . 7. Filter In some cases it is preferable to precipitate the protein and examine

THE TECHNICON SYSTEM

39

the filtrate, rather than use the dialyzer. The continuous filter performs this operation, and is often used for phosphatase assays. 3. I .8. Heating bath This constitutes the reaction vessel. It consists of coils of many feet of glass tubing which act as time-delay coils. The bath usually contains two separate coils which may be used in series or parallel. Coils of different sizes are available, so that the retention time may be varied using a constant flow rate from the manifold. For long incubation times, more than one heating bath may be used. There are many other devices in the Technicon system (e.g. an automatic digestor for the dermination of total nitrogen or phosphorus) and the reader is referred to the many brochures of the company for full details. Because of its widespread use, I will try to keep to the terminology used by Technicon and will use the term ‘flow system’ to describe the overall arrangement of the modules.

3.2. Automation of a simple-enzyme assay In the remaining chapters, I will often illustrate the various systems by examples from work from my laboratory. Many of the results were obtained in preliminary experiments during the development of the various systems and their scientific interest is of little relevance to this book. For this reason, I have sometimes simply given instrument readings rather than absolute values, and have included few details as to composition of reaction mixtures and source and preparation of enzymes. Most of the work has been with various ferricyanide reductases of yeast, and the enzyme preparations were usually homogenates obtained fiom yeast that had been disrupted with glass beads. In some experiments yeast mitochondria, kindly prepared by Dr. L. A. Grivell, were used. A useful way to introduce the reader to the Technicon system in particular, and continuous-flowautoanalysisingeneral, is to describe in detail a typical manual assay (of e.g. L(+)-lactate dehydrogenase of yeast). The enzyme is assayed by following the reduction of potassium ferricyanide by lactate at neutral pH. The Sublecr index p . 218

40

AUTOMATED ENZYME ASSAYS

ferricyanide absorbs strongly at 420 nm and none of the other reactants or products, including ferrocyanide, absorb strongly at this wavelength. In order to avoid complications due to the need for deproteinization, I will consider the simplest case in which a soluble extract is used. 3.2.1. The manual method The manual assay is as follows. A reaction mixture is prepared so as to give final concentrations, after addition of a suitable volume of enzyme, of 0.05 M potassium phosphate pH 7.4, 2.0 mM sodium L( +)-lactate and 0.4 mM K&(CN),. Enzyme is added to give a final concentration of 50 pg protein/ml, and after 10 min at 30 "C the fall in absorbance at 420 nm is measured. In detail, the actual assay proceeds in the following stages: 1. The stock solutions are prepared. (A stock solution is any solution that is used to prepare a reagent. The reagent may either be a suitably diluted stock solution, or may be a mixture of various stock solutions.) 2. The reagents are prepared by dilution of the stock solutions. The convenient stock solutions in this case are 1 M potassium phosphate¶ 50 mM lactate and 10 mM ferricyanide. Four reagents are prepared: A: 0.075 M potassium phosphate pH 7.4; B: 0.015 M sodium L(+)-lactate pH 7.4; C: 3 mM K&!(CN),; D: enzyme solution, 0.75 mg protein/ml. 3. The reagents, apart from the enzyme solution, are mixed to form a reaction mixture by adding 4 ml of (B) and 4 ml of (C) to 20 ml of (A). 4. Two test tubes each containing 2.8 ml of the reaction mixture are equilibrated in a water bath at 30 "Cfor 5 min. 5. Enzyme (0.2 ml of (D)) is added rapidly to each tube, the contents are mixed thoroughly, the E420 of the first tube is read immediately and the second is placed in a waterbath for 10 min. 6. The E420 of the second tube is read. 7. From the difference in values between the tubes and the known protein concentrations, the enzyme activity is calculated in terms of pmoles/min/mg protein. (The automation of calculations is discussed fully in ch. 8.)

41

THE TECHNICON SYSTEM

3.2.2. Dilution factors and fractionul volumes In order to clarify the subsequent discussion it is useful to tabulate the various initial and final concentrations and the volumes used (table 3.2). This table contains two new terms, the dilution factor and the fractional volume. These are used in automated systems, particularly for calculation of reagents required for a given reaction mixture. TABLE3.2 Example of a simple manual assay: reagents used. Component

Volume of Initial con- reagent centration (ml)

K phosphate pH 7.4 0.075 M Na L(+)-lactate 0.015 M K3Fe(CN)s 3.0 mM Enzyme 0.75 mg/ml Total

2.0 0.4 0.4 0.2

3.0

Final concentration

Dilution Fracfactor tional volume

0.05 M 2.00 mM 0.40 mM 50.0pg/ml

1.5

7.5 7.5 15.0

0.67 0.13 0.13 0.067 1.ooo

The dilution factor measures the extent to which a component in a stock solution is diluted in the final reaction mixture. It is: initial concentration of component final concentration of component

9

and is equal to: final volume of reaction mixture volume of stock solution added The reciprocal of this value is the fractional volume, and measures the contribution a particular reagent makes to the total volume of the reaction mixture. When these terms are applied to an automated system each reagent is identified by the line in which it is pumped. In the Technicon system, the line is the particular tube in the proportioning pump that is connected to the reagent. However, in a general sense it is any tube, probe or pump that connects a given reagent to the analytical system. For automation, we therefore use the terms SubjecI index p . 218

42

AUTOMATED ENZYME ASSAYS

line dilution factor and fractional line volume. For convenience these may be abbreviated to LDF and FLV respectively. 3.2.3. Adaptation to the autoanalyzer Let us now see how the above simple assay may be adapted to the autoanalyzer. A simple flow-system to perform the assay is shown in fig. 3.1. Phosphate and lactate are pumped into the system and mixed in a mixing coil (MC,). In order to produce a bubble pattern we introduce a stream of air at the same time. We thus have the arrangement: line 1 : substrate; line 2: buffer; line 3: air. Having mixed the substrate with the buffer, we now add the ferricyanide in another line (line 41, referred to as the co-factor line. Another mixing coil is used t o provide the complete mixed reaction mixture, ready for injection of the enzyme stream. Of course it would have been possible to prepare the mixture of buffer, substrate and co-factor in bulk, manually, before the assay, and in practice it is advisable to keep the number of lines

MC, -I//-

MC,

MC, ///P

1-

Substrate

2-

Buffer

3-

Air

4-

Co - f actor

5-

Enzyme

w*

Colorimeter Recorder

Fig. 3.1. A simple flow system for enzyme assay. D: de-bubbler; FC: flow cell; HB: heating bath; MC: mixing coils (MC3 is jacketed to operate at the temperature of the heating bath); W: waste.

THE TECHNICON SYSTEM

43

to a minimum. However, the system shown in fig. 3.1 is a useful one, since it may be used as a generalized manifold for many enzyme assays as discussed in detail in ch. 9. Enzyme is introduced through line 5 and the reaction starts as the enzyme mixes with the reaction mixture. This occurs in a jacketed mixing coil (MC,) which is at the temperature of the heating bath, making calculation of the time of incubation somewhat easier. The reaction mixture containing enzyme now enters the heating bath where the bulk of the reaction takes place. The effluent from the heating bath then passes directly to the de-bubbler. This is an Fshaped glass joint. Gas plus liquid are pumped into the lower bar of the F and the vertical limb is connected to the flow cell. The upper bar is connected to waste. The flow rates in the various pump-tubes are so chosen that the volume of fluid being drawn through the flow cell is slightly less than the volume of fluid entering. The excess fluid, plus gas-bubbles, rises to the upper bar of the F and hence to waste. The effluent from the flow cell is returned to the venous (or input) side of the pump. (The terms ‘venous’ and ‘arterial’ are useful in describing autoanalyzer systems. They remind us that the autoanalyzer is essentially a closed circulatory system operating under pressure caused by the pump, with some similarities to vascular systems. For example once we have opened the system to atmospheric pressure we can only obtain accurate flow rates if the fluid is returned to the venous side, just as it is impossible to control the flow from an open blood vessel.) The problems of bubbling and de-bubbling arise from the fundamental design of the autoanalyzer. The stream of bubbles prevents smearing of samples during their passage through long lengths of tubing (Skeggs 1957). The efficacy of the bubble train is shown in fig. 3.2, in which samples were run with and without bubbling. Some authors have described flow systems that do not use gas-segmentation. For example Hicks and Updike (1965) describe an automated procedure for estimation of lactate dehydrogenase. However, the sample traces presented suggested that the system had poor wash characteristics. The theory of sample interaction and wash is discussed in 9 8.2.7. Mixing coils in the Technicon system operate by the action of the Subiecr index p. 218

44

AUTOMATED ENZYME ASSAYS

bubbles rising and falling as the fluid passes through the coil. More rapid mixing may be achieved by so-called ‘jet-mixers’ in which fluid is forced through a tube with a series of constrictions that produce turbulent flow. The action of this device is shown in fig. 3.3. In most systems, the time spent in the mixing coil is about 1 min; mixing is usually complete within 30 sec. A1

A 2

0

A3

m

S -

0 0

B1

L

82

L

Fig. 3.2.Effect of gas-segmentation on the shape of thesample peak. The flow system in fig. 3.1. was used with a sampler attached to the substrate line (line 1). Water was placed in lines 2, 4,and 5, and three identical samples of 2 mM potassium ferricyanide were placed in the sampler, with water in alternate cups. Al-A3: peaks obtained with air in line 3,giving the usual stream of air-bubbles (air-segmentation) ; Bl-B3: peaks obtained with water in line 3.There was no stream of air bubbles. However, with the type of sampler used (sampler I) there was a pulse of air between samples caused by the sample crook being in air; C143: the sample probe was dipped manually into the samples, with rapid transfer so as to eliminate any air bubbles in the stream; S: steady state with sample probe in 2 mM potassium ferricyanide. (Note that poor sampling is shown by failure to approach the steady state value, and spike-shaped sample peaks.)

THE TECHNICON SYSTEM

45

Inner segments (0.035)

Fig. 3.3. Design of jet mixer. Fluid flowing through the mixer is confronted with repeated sudden alterations in tube diameter. This produces marked turbulence and hence good mixing. (From Pitot et al. (1968), by courtesy of the authors.)

The requirement for a bubble train plus the rather stringent requirements of the de-bubbler cause some difficulties in the design of more complex manifolds. This is because few sensors, if any, can operate on a stream of fluid interrupted intermittently by bubbles. For example, a continuous-flow spectrophotometer would give a very irregular trace that would be impossible to interpret. In fact, if the waste return from the de-bubbler is not adjusted correctly occasional bubbles may pass through the colorimeter, spoiling the trace. As a result, in order to use a sensor one must de-bubble. This usually involves an additional waste line to the manifold, hence increasing its complexity. Also, once de-bubbled, smearing of the sample occurs rapidly and the lead from the de-bubbler to the sensor must be as short as possible. Any further operations on the stream must be done on a re-bubbled stream, which entails returning the unsegmented line to the venous side of the pump. During this process smearing can occur, with loss of resolution. The re-bubbling requires another gas line, so that insertion of an extra sensor into the system requires a new waste line and gas line, and also results in possible loss of sample resolution. A method for making measurements on an intermittent stream of liquid and gas would greatly simplify many Technicon systems. Recently, Technicon have introduced a device for producing a very regular bubble pattern. A small clamp rises and falls over the gas inlet line at regular intervals. With such a regular pattern it might be possible to have a time-controlled sensor which would only make measurements when fluid was passing through the system. Having explained the general design and principle of operation of Subject index P. 218

46

AUTOMATED ENZYME ASSAYS

the flow system, I will now consider the choice of pump tubing. In the manual assay we mixed buffer, substrate, co-factor, and enzyme solutions in the proportions by volume of 2.0 :0.2:0.2 :0.2. We could try to find pump-tubing that had precisely these relative flow rates, i.e., one tube that delivered 2 ml/min and three tubes that deliver 0.2 ml/min. However, it is usually better to have flow rates in the various tubes nearer to each other than these ratios. This is because fluid injection from a slow stream into a fast stream is often imperfect and can lead to distortions in flow. Also we have to allow for the gas line and for loss of fluid at the de-bubbler. It is rather difficult to select an exact time of incubation. The reaction vessel has a fixed volume and with the standard pump one cannot vary the flow rate continuously. The retention time is determined by the ratio of vessel volume to flow rate (see Q 8.2.2) and hence cannot be varied continuously either. (This is possible, however, with a variable speed pump, as discussed in Q 9.2.1.) Fortunately, an important advantage of automated systems resulting from the high degree of reproducibility of the operations is that actual time of incubation is generally not critical, provided that it is reproducible. In our example, however, we will try to obtain an incubation time fairly near that used in the manual assay. To do this we may either use a total flow rate of about 2 ml/min with one coil in the heating bath, or about 4 ml/min with two coils. It is often difficult to choose between the alternatives of a slower flow with a smaller vessel and a faster flow with a larger vessel. In general, the smearing of samples is less if the length of tubing in the delay coils is kept as small as possible, but lower flow rates produce less eficient washing of joints and connections. The ideal system would have a fast flow rate and a small retention volume. However, this will reduce the amount of reaction occurring, and may lead to difficulties in measuring the reaction because of insensitivity of the sensor. In our case we will use the faster flow rate with two coils. Best bubble patterns are obtained with the gas line pumping 4 to 3 of the total fluid pumped, so that in our example a convenient ratio would be: air 1 ml/min; total liquid 3 ml/min. For convenience of design we will use lines with the following flow rates: buffer line 1.2

THE TECHNICON SYSTEM

47

ml/min; substrate line 0.6 ml/min; co-factor line 0.6 ml/min; and enzyme line 0.6 ml/min. Having established these arbitrary line volumes we now work back to calculate what reagents may be connected to these lines to give a reaction mixture of the required final concentrations of each component. These reagents are conveniently called line reagents to distinguish them from the stock solutions from which they are prepared (see #9.2.5,9.3.5,9.3.6, and appendix IV). We now prepare a table similar to table 3.2 except that we now use line dilution factors (LDF) and fractional line volumes (FLV). (Note that for the purposes of calculation of reaction mixture concentrations, the gas lines are ignored. This is permissible since generally the gas does not enter into the composition of the reaction mixture. If it did, for example in C02/HC03- systems, due allowance would have to be made for this.) It is useful to specify line numbers by a subscript so that LDF, and FLV, are the values for the nth line. (A detailed treatment of the calculations is given in § 8.2.3.) Here, one may merely note that the final concentration of a component is equal to the product of its concentration in the line reagent and the fractional line volume of that line. Table 3.3. shows the arrangement of pump tubing for automation of our example. This system may be used for any assay in which buffer, substrate, co-factor, and enzyme are mixed and incubated for 5-10 min (i.e., with one or two coils in the heating bath). To prepare the appropriate line reagents all we have to do is multiply the required concentrations by the appropriate LDF values. For example, in an assay for malate dehydrogenase, the final reaction mixture consists of 0.05 M Tris/HCI, p H 7.6; 15 mM potassium oxaloacetate; 0.2 mM NADH; and 15 ,ug/ml of protein. The automated assay would have 0.125 M Tris/HCl in line 1,0.075 M potassium oxaloacetate in line 2, 1.0 mM NADH in line 4 and 75 pg/ml protein in line 5. More complex reaction mixtures are made by adding other reagents to the buffer or co-factor lines providing that the mixtures are compatible and stable. Thus it is more convenient to use a standard generalized assay system and alter the concentrations of the line reagents than to use a different pumping scheme for each assay. (A table of ratios of pumping rates Subject index p. 218

48

TABLE3.3

Reagents and pump tubing for automatioil of assay in table 3.2. (Flow system in fig. 3.1 used, with standard speed pump.)

Fractional line volume (FLV)

K phosphate Na L(+)-lactate Air KsFe(CN)e Lactate dehydrogenase

0.125 M 10.0 mM

1.2 0.6 1 .O 0.6 0.6

2.5 5.0

0.4 0.2

2.0 mM 0.25 mg/ml

Waste Total (excluding gas and waste lines)

2.0 3.0

-

-

5.0 5 .O

0.2 0.2

-

-

1 .O

AUTOMATED ENZYME ASSAYS

Line dilution factor (LDF)

Buffer Substrate Gas Co-factor Enzyme

Final concentration in reaction mixture

Line flow rate (ml/min)

6

Initial concentration of line reagent

5

Component pumped by line

4

Description

Line no. 1 2 3

THE TECHNICON SYSTEM

49

TABLE 3.4 Ratios of pumping rates with technicon tubing.* B/A B: 0.32 0.42 0.60 0.80 1.00 1.20 1.60 2.00 2.50 2.90 3.40 3.90 A ~~

0.32 0.42 0.60 0.80 1 .00 1.20

I .60 2.00 2.50 2.90 3.40 3.90

~

1.00 0.76 0.53 0.40 0.32 0.27 0.20 0.16 0.13 0.11 0.09 0.08

* See table

1.31

1.00 0.70 0.52 0.42 0.35 0.26 0.21 0.17 0.14 0.12 0.1 1

1.87 1.43 1.00 0.75 0.60 0.50 0.37 0.30 0.24 0.21 0.18 0.15

2.50 1.90 1.33 1.00 0.80 0.67 0.50 0.40 0.32 0.27 0.23 0.20

3.12 2.38 1.67 1.25 1.00 0.83 0.62 0.50 0.40

0.34 0.29 0.26

3.75 2.86 2.00 1.50 1.20 1.00 0.75 0.60 0.48 0.41 0.35 0.31

5.00 3.81 2.67 2.00 1.60 1.33 1.00 0.80 0.64

6.25 4.76 3.33 2.50 2.00 1.67 1.25 1.00 0.80 0.55 0.69 0.47 0.59 0.41 0.51

7.81 5.95 4.17 3.12 2.50 2.08 1.56 1.25 1.00 0.86 0.73 0.64

9.06 6.90 4.83 3.62 2.90 2.41 1.81 1.45 1.16 1.00 0.85 0.74

10.62 8.09 5.67 4.25 3.40 2.83 2.12 1.70 1.36 1.17 1.00 0.87

12.19 9.28 6.50 4.81 3.90 3.25 2.44 1.95 1.56 1.34 1.15 1.00

3.1 for colour code. Determine the required ratios of reagents e.g.

1.0:0.5:0.1:0.05. Select a convenient pumping rate for 1.0 and read-off the pump

tubing giving the nearest ratios. Thus if 3.90 ml/min, is chosen for 1.0, the nearest tubing to give the above ratios are 3.90 ml/min, 2.00 ml/min, 0.42 ml/min, and 0.32 ml/min. (Tubing of flow rate less than 0.3 ml/min is not included in table because of variation in observed flow rates.)

is presented in table 3.4.) There is usually nothing fundamental in the relative volumes of reagents that are used in manual assays, and they are often dictated by simple factors such as the availability of standard pipettes. It should be stressed that the above example, although dealing specifically with the Technicon system, is applicable to all automated systems, whether the reagents are mixed by proportioning pumps, individual pumps, automatic pipettes or other devices. It is still necessary to set the instrument in such a way as to deliver required volumes from different reagent bottles and unless the device exactly copies manual methods the above treatment is required.

Subiecr index P. 218

50

AUTOMATED ENZYME ASSAYS

3.3. The sampling process The sampling process is fundamental to all automatic assay systems. I n the Technicon system the sampler module introduces components into the stream in a repetitive but discontinuous way. An examination of its characteristics is relevant to the problem of sampling in general. The sampler may be used simply to introduce different samples of the same enzyme into the analyzer (‘enzyme sampling’), to test a mixture of enzymes with different substrates (multiple-enzyme analysis) or to make various additions to the reaction mixture during an assay (e.g. addition of inhibitors or activators). Recently there has been a tendenC

t

J

0 N P

0

0

w

3 !J

0 N

4

d

Time-

Fig. 3.4. Analysis of enzyme samples. Four different preparations of yeast L(+)lactate dehydtogenase were placed in sample cups, using the flow system in fig. 3.1 with a sampler attached to line 5. The reaction was followed by fall in E420 on reduction of ferricyanide.

THE TECHNICON SYSTEM

51

cy for manufacturers of automatic spectrophotometers to develop automatic sample changers to be used with their equipment (see appendix 111, p. 185). Usually they are designed simply to introduce the sample directly into the flow cell for measurement, and there is no provision for mixing reagents between sampling and reading in the sensor. However, such samplers could very easily be adapted for automated enzyme assay by using a suitable multi-channel pump and mixing chamber (see appendix 111, p. 189, for details of such pumps). The action of a simple enzyme sampler may be seen as follows. Line 5 in the manifold shown in fig. 3.1. is connected to a sampler operating at 40 cups/hr with a sample/wash ratio of 2: 1 (i.e., 1 min of sampling is followed by 30 sec of wash). Several enzymes placed in the cups of the sampler will then produce several peaks (fig. 3.4). Increasing the sampling speed increases the sharpness of the peaks, until eventually they do not reach the ‘steady-state’ level. There is an element of judgement as to what constitutes the best sampling rate. Although it is true that fast sampling can result in values less than the steady state and also in greater contamination between samples, these errors may be acceptable provided that they are reproducible. The absolute rate of sampling is an important factor in automation, and it may be better to accept a certain loss in precision in order to improve the rate. The theory of sampling is discussed in more detail in 0 8.2.7. During the assay, reagents pumped between samples, i.e., in the wash period, are wasted. A significant saving of expensive reagents, substrates or co-factors may be achieved if they are sampled in phase with the enzyme samples. Thus Hopkinson and Lewis (1967) developed an automated system for phosphoglucomutase in which a double sample probe was used, one for the enzyme sample, and one for the expensive substrate, glucose-1-phosphate. Discrete-sample analyzers (see 0 4.2) are really an extension of this approach, in that all the components of the reaction mixture are introduced into the reaction vessel in a discontinuous fashion, so that there is no flow of reagents between enzyme samples. The question of cost of reagents is not unimportant, particularly for large automated systems, and the use of multiple-sampling is certainly useful in reducing these costs. Subject index p . 218

52

AUTOMATED ENZYME ASSAYS

The shape of the sample peak is an interesting index of the performance of the analytical system. In a perfect system with no smearing or cross-contamination, the peak would be a perfect square-topped curve. However, in practice the upswing and downswing parts of the curves are spread in a manner discussed in more detail in 0 8.2.7. If we simply introduce a marker substance (e.g. potassium ferricyanide) into the sampler we would obtain a typical curve of the shape shown in fig. 3.5a. If an enzyme is sampled, this would correspond to the rise and fall in enzyme concentration. However, unless the autoanalyzer is measuring the enzyme directly by some property (such as a characteristic absorption band) the actual record obtained will be that of enzyme actioity rather than absolute concentration. Since in general the activity should be proportional to concentration, the curve with the enzyme should have the same sample shape as that with the marker. However, this would not be so if there were deviations from linearity in the assay. For example, let us assume that the relationship between observed rate and concentration is as in fig. 3.5b. The actual rate observed on the analyzer with a sample of shape as in fig. 3.5a would be that shown in fig. 3 . 5 ~ .It can be seen that there is an apparent (a )

(b)

~

'0

10

.

n

1

Time (min)

2

(C

p 10

1

~

n 1 2 Enzyme concent r a t ion

OO

.I Time ( m i d

Fig. 3.5. Distortion of sampling peak due to lack of proportionality to enzyme concentration. (a) Typical sampling curve for rise and fall in enzyme concentration, assuming a half-wash time of 10 sec (see ch. 8). (b) Non-linear relation between observed activity andenzymeconcentration. (c) Sampling peakexpected with(b). (The units of enzyme activity and concentration are arbitrary, and all values are hypothetical.)

53

THE TECHNICON SYSTEM

broadening of the curve, and this would result in less resolution between samples. Because the pen moves rapidly in the upswing and downswing of the sample peak, there have been few precise studies on the shape of the peak during enzyme sampling. However, much useful information may be derived from such a study. For example precise comparison of the traces with markers and enzyme catalyzed reactions, perhaps with faster recorders, or deliberately poor sample resolution, would give an automatic record of the relation between rate and enzyme concentration. The sampler I1 module has facilities for refrigerating the enzyme samples (Tappel 1965) and for stirring enzyme suspensions before sampling, so that such studies are quite feasible. In multiple-enzyme analysis, the substrate is placed on the sampler table, and aspirated into a continuous stream of enzyme. Again, close examination of the shape of the peak could well yield much information. Fig. 3.6 shows three curves as follows: (a) a typical curve with a marker, as in fig. 3.5a; (b) a hypothetical plot of substrate concentration against enzyme activity, using the usual MichaelisMenten relationship; and (c) the expected curve if the substrate is sampled over the range shown in (b). Again distortion of the curve occurs, and in this case precise comparison of (a) and (c) would

[k c

0

c

L c

n

oo

1

Time (min)

-2 0 0

8

16

Substrate concentration

Fig. 3.6. Distortion of sampling peak due to Michaelis effect. (a) Typical sampling peak for rise and fall in substrate Concentration, as in fig. 3.5a. (b) Hypothetical plot of enzyme activity against substrate concentration (Km = 4 arbitrary units, Vmsx = 10 arbitrary units). (c) Sampling peaks expected with (b). (Units for all scales arbitrary, and all values hypothetical.) Subject index P. 218

54

AUTOMATED ENZYME ASSAYS

enable one to calculate curve (b) and hence the Michaelis constant and maximum velocity of the reaction. It is clear, therefore, that precise study of the shape of the sample peak could well yield fruitful results.

3.4. Interference by protein I will now discuss the problem of interference by protein in the enzyme assay. In general it is preferable not to remove protein, but to work under conditions in which the enzyme is sufficiently active for the concentration to be kept low during the assay. However, sometimes this is not possible. The Technicon system uses two modules, the dialyzer and the continuous$lter. Recently a discontinuous centrifuge for deproteinizing blood has been described by Quickfit & Quartz (see appendix 111, p. 181). In this the sample to be centrifuged is introduced into the drum centrifuge. After centrifuging a probe removes the supernatant fluid and the sediment is sucked off to waste so that the centrifuge is ready for the next sample. 3.4.1. Continuous dialysis The Technicon dialyzer (fig. 3.7) has a suitable membrane clamped between plates over which the protein flows. There are four streams, two input and two output. The donor stream passes material across the membrane to the acceptor stream so that there are input and output donor streams and input and output acceptor streams. (I have avoided the term ‘dialyzate’ as this is ambiguous.) For example, to dialyze a solution of serum albumin and glucose against phosphate output

Input

t-

Fig. 3.7. Principles and proposed nomenclature of Technicon dialyzer. The dialyzed material passes from the donor to the acceptor stream.

THE TECHNICON SYSTEM

55

buffer in order to estimate glucose in the absence of protein, the input donor stream would contain the glucose-protein solution and the input acceptor stream phosphate buffer. Unfortunately, the dialyzer has a low efficiency, usually between 10 and 20%. Thus, although the output acceptor stream will be free of serum albumin, the output donor stream will still have a high glucose concentration, With 10% efficiency, the output acceptor stream will have about 1/10 of the glucose concentration of the output donor stream. This makes calculations of absolute enzyme rates with the dialyzer somewhat difficult. A disadvantage of the dialyzer is that it may alter its properties during the course of the assay. The efficiency of dialysis is affected, for example, by the relative ionic strengths of donor and acceptor streams. If these were to alter during the assay, for example by oxidation of an organic ion to C 0 2 and water, one may have different rates of dialysis as the reaction proceeds. For the dialyzer to work efficiently there must be good bubble patterns in the donor and acceptor streams. It is also preferable if the donor and acceptor streams have fluid segments that are roughly of the same length. Finally, the dialyzer has fine grooves in the dialyzer plate for the passage of fluid. If the enzyme solution is not perfectly free from debris, sediment accumulates particularly at the input nipples. This leads ultimately to complete ‘thrombosis’ with a dramatic bursting of the input leads from their nipples. 3.4.2. Continuousfiltration For the above reasons, the continuous filter is often preferable. Its design and method of operation are shown briefly in fig. 3.9. Fluid containing protein and low molecular weight material is passed into the mixing chamber where it is agitated with a stream of precipitant, such as 5% trichloroacetic acid. The mixture then drips onto a moving strip of filter paper and some of the filtrate (F) is returned to the analyzer. The filter differs from the dialyzer in that the concentration of low-molecular weight substances is the same on both sides of the filter. In calculation of enzyme rates, therefore (ch. 8), one has merely to allow for the dilution caused by the addition of the precipitating Subiecl index D. 218

56

AUTOMATED ENZYME ASSAYS

1

1

Substrate Buffer Co-factor Enzyme Buffer G a5

Colorimeter Recorder

Fig. 3.8. Flow system for removal of protein by dialysis after enzyme sampling. After the reaction has taken place in the heating bath the reaction mixture is dialyzed against an air-segmented stream of buffer. The output acceptor stream (see fig. 3.7) is passed to the sensor. D: de-bubbler; FC: flow cell; HB: heating bath; JMC: jacketed mixing coil; S: sampler; W: waste. 7

JMC

MC

MC

Substrate Buffer Co- factor

Enzyme Mixer

Colorimeter

Trichloroacetic acid

Recorder

Fig. 3.9. Flow system for removal of protein by filtration after enzyme sampling. The reaction mixture is mixed with a de-proteinizhg agent and passed through the continuous filter to the sensor. (Symbols as in fig. 3.8.)

THE TECHNICON SYSTEM

57

agent. The filter has an effect on the sharpness of the peak, and assays run with it generally have to operate at slower sampling rates. Heinicke et al. (1967) present a useful table of the properties of different filter papers and their efficacy in removing protein precipitates. Burns and Lazer (1965) used the filter to assay polynucleotide phosphorylase and Hopkinson and Lewis (1967) to assay phosphoglucomutase. A flow system for multiple phosphatase analysis using the continuous filter has recently been developed in our laboratory (Jowett and Roodyn 1969). To conclude this chapter, two analytical systems are given for the simple assay discussed at the beginning of the chapter, with enzyme sampling and removal of protein. In the first (fig. 3.8), the sampler is connected to the enzyme line, and enzyme samples are placed in the sampler cups. The effluent from the heating bath is passed into the input donor stream of the dialyzer. The input acceptor stream is 0.05 M phosphate buffer, and after dialysis the output donor stream is allowed to go to waste and the output acceptor stream is passed through the colorimeter, and thence to waste. The arrangement with the continuous filter is similar (fig. 3.9) except that the input acceptor stream contains 5% trichloroacetic acid. This chapter has not listed different detailed assays, but has tried to give a general picture of the processes involved in automating relatively standard enzyme assays. The adaptation of manual methods to automatic systems is not always as simple as the above account may indicate. For example, reagents may not behave the same way in the coils and plastic tubes of an autoanalyzer as they do in the test tube. Schwartz (1967) gives some examples of these problems. For example, in an assay for phosphohexose isomerase, veronal acetate buffer precipitated when acid was added and blocked the manifold. Use of Tris buffer instead solved the problem. In the manual assay the resorcinol formed was dissolved in ethanol. In the autoanalyzer this solvent boiled and the resulting vapour interrupted the flow and gave irregular traces. Substitution by ethylene glycol solved the problem, although it gave a lower colour yield. I have found that use of aminonaphthol sulphonic acid in phosphate determinations causes Subject index p . 218

58

AUTOMATED ENZYME ASSAYS

blockage of the lines, which is avoided by using ascorbic acid as a reducing agent. One must also consider the possibility of surface denaturation of enzyme during the assay. It is also difficult to automate methods in which the enzyme preparation is very viscous, for example with bacterial lysates containing much DNA. It is therefore necessary to consider each case on its own merits, but in the light of the general principles indicated above. (A detailed example of an automated enzyme assay is given in 0 9.4.)

CHAPTER 4

Interrupted -flow and disCrete-sampling systems

4.1. Interrupted-flow systems We have seen in ch. 2 how the semi-automatic methods rely on manual loading of the autosensor. Interrupted-flow methods perform this automatically, i.e., the reaction mixture is prepared, mixed with enzyme and pumped into the sensor, usually the flow cell of a recording spectrophotometer. Once the reaction mixture is in the sensor, the flow of fluid is stopped and the course of the reaction is followed on a stationary reaction mixture. After a predetermined time, flow of fluid recommences, the sensor is washed free of old reaction mixture, new reaction mixture plus enzyme is introduced and the next assay is performed. The great advantage of such a system is that true initial rates may be measured. This is shown in fig. 4.1. In (a) four enzyme reactions are followed in sequence by filling the autosensor with the first reaction mixture plus enzyme, monitoring the reaction for 4 min, washing out the reaction mixture, filling with the next reaction mixture plus enzyme and so on until all four enzymes are measured. Thus a complete progress curve is obtained in each case for a period of 4 min each. In (b) the same reactions are followed in a continuous-flow system. Enzyme and reaction mixture are fed in continuously and the reaction is measured 1 min after enzyme was mixed with substrate. The four values obtained are given by the vertical bars and are seen to fall within the linear regions of all four progress curves in (a). However, if the continuous-flow system is arranged so that the reaction is measured 4 min after mixing, as in (c), only two enzymes (E, and E4) 59

Subject index P. 218

60

._ 73 c

(51

m

a,

LK

AUTOMATED ENZYME ASSAYS

4

Fig. 4.1. Advantages of monitoring progress curve compared to single time-points assays. (a) Four enzymes assayed by interrupted-flow method. (b) The same enzymes assayed by a continuous-flow system with a single time of incubation and measurement of 1 min. (c) Continuous-flow system with single time of incubation and measurement of 4 min.

are measured in the linear region, assays for E, and E, giving low relative values, particularly for E,, Unfortunately, the advantage of being able to determine the initial rate is offset by the relative slowness of interrupted-flow methods if the reaction cannot be measured in a short incubation time. For example, if it takes 1 rnin to prepare the reaction mixture, and a reasonable reading is obtained after only 2 rnin of incubation, the total time for 50 samples is 2 hr 30 rnin using interrupted flow. With a continuousflow method that also requires 1 min to prepare the reaction mixture and has a 2 rnin retention time, the total time of assay for 50 samples is only 53 min (assuming a sampling rate of l/min). The time gained by the continuous-flow method is due to the fact that the analytical system is not halted during the assays. The gain in time with continuous-flow assay becomes much greater if the sample activity is not very high, or the method of detection not very sensitive. If 20 min are required to obtain a valid reading, the interrupted-flow method above

INTERRUPTED-FLOW AND DISCRETE-SAMPLING SYSTEMS

61

will take 17 hr 3 min for 50 samples. With continuous flow, 21 min will elapse before the first sample is measured, but thereafter samples will be obtained at the rate of one per min, so that the total time will be only 1 hr 11 min! The rate of analysis with the interrupted-flow method is improved somewhat if more than one sample can be read at a time (e.g. if a spectrophotometer with an automatic cell changer is used with 4 cuvettes) but in general the method can only operate a t a rate of analysis comparable to continuous-flow methods if the time of incubation is small. Where the actual rate of analysis is not a critical factor, clearly measurement of the entire progress curve on a stationary reaction mixture has great advantages. Continuous monitoring of enzyme activities is difficult to perform with interrupted-flow systems because of the intermittent flow of enzyme. As a simple example of the method, an interrupted-flow system using two pumps is shown in fig. 4.2. The reaction mixture is pumped into a temperature-controlled reaction vessel, the enzyme is added manually, and the mixture is then pumped out from the vessel through a continuous-flow colorimeter. A typical trace is shown in fig. 4.3. The

-

Pump 2

n

W -

II

Enzyme (add manually)

0.8ml/min

I

Pump1

n

vessel

Fig. 4.2. Semi-automatic interrupted-flowsystem. The assays are performed by the following sequence of operations: (1) Turn on pump 1 for 50 sec to fill jacketed reaction vessel with reaction mixture. (2) Turn on pump 2 to flush flow cell with reaction mixture for 20 sec. Turn on recorder. (3) Remove stopper of mixer vessel and rapidly blow in enzyme from a micropipette. (4) Follow reaction for 2 min. (5) Turn off recorder, wash out reaction vessel. Return to (1) for next assay. Subiecr index p . 218

62

AUTOMATED ENZYME ASSAYS

I 0 [ L C U

4 4

h

4

0

OD

4

f N 0

LL'

1 rnin

-

Time b Fig. 4.3. Trace obtained with semi-automatic interrupted-flow system of fig. 4.2. RM: addition of reaction mixture for assay of L(+)-lactate dehydrogenase. (Reading corresponding to I pmole/ml calculated by calibrating recorder with G ,electron acceptor used in the assay.) Enzyme: standard solution of K ~ F ~ ( C N )the suspension of yeast mitochondria kindly prepared by Dr. L. A. Grivell. Numbers refer to pl of enzyme added.

system has the advantage of not using the colorimeter cuvette as a reaction vessel. It was, in fact, operated manually by following a sequence of times on a stop-watch. However, it is a relatively simple matter to connect the pumps to suitable timers which would control their operation. One of the difficulties of these systems is surging of the reaction mixture caused by suddenly stopping its flow. In particular, surge in the cuvettes can produce erratic readings. Pollard (1964) has described an interrupted-flow system in which a simple solenoid-operated clamp stops the flow into the cuvette. The most detailed work with interrupted-flow systems has come

INTERRUPTED-FLOW AND DISCRETE-SAMPLING SYSTEMS

63

from Pitot and his associates. Pitot and Preis (1964) described a system called a ‘combination unit’ that consisted of a Gilford 2000 multi-sample absorbance recorder (see appendix 111, p. 183), a Beckmann DU monochromator with thermostatted chamber, a Technicon large sampler and a Technicon proportioning pump. The system is described in detail by Pitot et al. (1965, 1966). The enzyme samples to be assayed are placed in the sampler table and various lines to the pump feed in buffer, co-factor, and substrate. According to a predetermined time schedule, the reaction mixtures are mixed with enzyme and pumped into the flow cells of the Gilford 2000 system. The pump and sampler are then stopped by relays controlled by a timing unit and the reaction is allowed to proceed in 4 cuvettes. After a suitable time, the pump and sampler are started again and new enzyme samples are introduced. The recorder trace obtained resembles that obtained by semi-automatic methods and the system allows inspection of the entire progress curve and selection of values from the linear region of the curve. It has been applied to the assay of many enzymes (see appendix I). Recently Pitot et al. (1968) have described a more complex system (fig. 4.4) with facilities for automatic data processing.

4.2. Discrete-sampling systems Recently, several automatic systems have appeared on the market which are intermediate between interrupted-flow and continuous-flow analyzers. One of the earliest of these was the ‘Robot Chemist’ and examples of its use in enzyme assay are given by Morgenstern et al. (1965c, 1966a, 1967), and Klein et al. (1966). Although the various discrete-sample analyzers differ greatly in the physical arrangement of their component parts and their overall complexity, they are all based on the following principle: A train of reaction vessels moves continuously through the incubation bath. The train may be linear or circular, with samples being transported either in linked chains of vessels, or more commonly on circular turn-tables. At various points in the train, automatic pipettes introduce reagents and enzyme into Subject index P. 218

64

AUTOMATED ENZYME ASSAYS 4

I

+

-

4

.

7

1

-

Upper level Lower level

1

2’

~

ervoir

Fig. 4.4. Manifold for multiple assays using four sample lines. Reagents plus enzyme are pumped from the reservoir and sampler on the right and mixed by ‘jet mixers’ which have a series of constrictions to produce turbulent flow. They are pumped into the flow cells of the spectrophotometer for monitoring of thereactions, and subsequently to waste. ‘Upper and lower levels’ refers to positions of lines in the proportioning pump. (From Pitot et al. (1968), by courtesy of the authors.)

the reaction vessels, with stirring usually effected by the swirling caused by injection of fluid. At a later point in the train, sample probes aspirate the reaction mixture into the sensor to measure the reaction. In some systems, the reaction vessel itself passes into the sensor and acts as cuvette. However, more often the sample probes are connected to a continuous-flow device in the sensor. After measuring the reaction, the reaction mixture is either passed to waste, or in some systems returned to the train for further processing. The volumes of reagent and enzyme are determined by settings on the automatic pipettes and it is usual to have pipettes in the 100 pl range for enzyme samples, and 1-5 ml range for reagents. The incubation time is determined by the

65

INTERRUPTED-FLOW AND DISCRETE-SAMPLING SYSTEMS

Reagents

- - - /

-

.

I

--I ,

-

- - --

Samples t r a i n

- - -

-

and /or printer

- -

I

- -

React ion t r a i PI Incubation vessel

Fig. 4.5. General principles of discrete-sampling analysis. A linear or circular train of enzyme sample cups moves past an automatic pipette PI which aspirates a suitable volume (A), moves its position (B), and delivers the enzyme sample (C) to a train of reaction vessels. After a suitable time of temperature equilibration, reagents are introduced by one or more automatic pipettes (Pz)and the reaction is started. A final probe (P3)transfers some of the reaction mixture into a continuousflow sensor. In some analyzers, the cups in the reaction train are optically standard and act as cuvettes of a colorimeter, so that no transfer a t P3 is necessary. Usually the sample and reaction trains are separate but sometimes they are on a single turn-table. The times between sampling, addition of reagents, and measurement of reaction are determined by the distance between probes and rate of movement of trains. (See appendix 111 for details of specific instruments.)

distance between the last pipette to produce the reaction mixture (usually the enzyme pipette) and the sensor, and of course by the rate of movement of the train. The principles of discrete sampling are shown in fig. 4.5. Usually, the discrete sampler gives a single time of incubation. However, the time course of the reaction could be followed in principle by placing sensor probes along the train, or by mixing enzyme and substrate rapidly just before the sensor probe and following the course of the reaction while the reaction mixture is in the sensor. It is still somewhat early to assess the merits of discrete-sample systems as compared to the older continuous-flow systems, particularly of Technicon. The various instruments vary greatly in design, and Subject index I . 218

66

AUTOMATED ENZYME ASSAYS

some are not specifically intended for enzyme assays. In general the method has the theoretical advantage of greatly reducing, if not eliminating the problem of inter-sample contamination, provided that enough sample is used, so that higher sampling rates are possible (e.g. up to 300/hr). In addition, there is less danger of surface denaturation of enzymes since the surface over which the enzyme flows is reduced. It is rather surprising that very few authors have reported significant differences between results with the Technicon system and manual methods. This would indicate that the bubble pattern is not as great a source of surface denaturation as expected. However, there have been some reports of difficulties with the Technicon system. An extreme case was the report of Kirk (1967) that glutamotransferase appeared to be strongly and specifically adsorbed onto the glass surfaces of the autoanalyzer. However, discrete-sampling systems have some lengths of tubing through which the enzyme must flow, so that the problem may still exist there. The absence of the bubble pattern makes for easier monitoring of reaction mixtures, but the sensor probes must then be reasonably near the reaction vessel, thus making the instrument less flexible. This lack of flexibility probably is the major disadvantage of the systems so far produced. Apart from multi-channel systems, all parts of the train must be exposed to the same incubation conditions. There are some systems for transferring reagents from one turn-table to another, but the instrument is specifically engineered for this purpose. Some discrete samplers use disposable reaction vessels; others use a train of vessels that are washed in a ‘tube laundry’ of some sort. Here there is a danger that an occasional vessel may become contaminated. Since the blank assay would be carried out in another such vessel, it may be difficult to detect random contamination. Most of the discretesample analyzers do not give a continuous record of the progress of the reaction in the vessel, or indeed of the flow of reaction mixture into the sensor. Usually the sensor is connected to some digital output device and the value is printed out. This is a rather serious limitation in routine analytical work, since faulty loading of a tube, or even an empty tube, would give a spurious value that may not be obvious. A

INTERRUPTED-FLOW AND DISCRETE-SAMPLING SYSTEMS

67

half-empty sample cup in the autoanalyzer, however, would give a narrower peak than usual. The problem of drift and blanks is perhaps greater in the discrete-sample system. With continuous-flow systems it is possible to have continuous assessment of blanks and base line drift with the actual reagents and reaction vessel used, since all samples use the same stream; in discrete systems an error that occurred with one reaction vessel may not be repeated with another. From the point of view of instrument design and maintenance, discretesample systems have more moving parts than the continuous-flow ones and hence a greater risk of instrument failure. Such failures may be more difficult to detect than in the autoanalyzer. For example, in the assay described in 4 3.2 changes in the pumping rate of any of the lines is immediately detected by a change in the base-line at 420 nm. A discrete-sample analyzer giving digital values may not give such a clear and immediate indication. In spite of all these difficulties, there is little doubt that the discretesample analyzer will be of great value, particularly in the analysis of samples by routine and well established methods. Problems in calibration, detection of blank drift and occasional failure of components in the train are not insurmountable. Perhaps the greatest problem is the lack of flexibility, but again perhaps improved modular construction will be of help. Details of discrete-sampling systems are given in appendix 111.

Subject index P. 218

CHAPTER 5

Single-enzyme analysis

In this chapter 1will discuss in more detail methods in which a single enzyme-catalyzed reaction is studied. Most of the examples will be with continuous-flow systems, but the arguments apply in many cases to all types of analyzer. The usual use of single-enzyme analysis (S.E.A.) is to measure the activity of a number of samples, for example in discrete serum samples, or in the effluent from a column. Another common use is to employ the reaction to estimate substrate, e.g., the determination of alcohol by alcohol dehydrogenase.

5.1. Molar conversionfactors and enzyme units The operation and calibration of enzyme analyzers is affected to some extent by the way activities are finally calculated. As mentioned in ch. 1, the most acceptable method is to express results as pmoles of substrate transformed per min and for specific activities as pmoles per min per mg protein. In order to do this, certain absolute measurements have to be made on the instrument, perhaps the most difficult of these being a precise measure of incubation time. However, it may be acceptable to use arbitrary units. For example, one may simply say that an instrument reading of 1.0 corresponds to one enzyme unit. For this unit to have any meaning, however, we would have to establish that theie was a linear relationship between instrument reading and enzyme concentration. This could be done by analyzing a range of enzyme concentrations introduced either manually or with a suitable 68

SINGLE-ENZYME ANALYSIS

69

gradient-making device (see $8 6.3 and 7.5). If the curve so obtained were non-linear, we would have to use it as a ‘calibration curve’ for each assay and calculate values by graphical or numerical interpolation. An improvement on the use of totally arbitrary units is to use so-called ‘standard’ enzymes which are commercially available and sometimes used in clinical enzymology. Since even the most stable enzymes may be inactivated under certain conditions, it is bad practice to assume that the value on the label is correct. If facilities are available, the standard should be checked either by the usual manual assay or by a semi-automatic method and a calibration curve constructed in terms of the true activity of the standard. In general the use of ‘standard’ enzymes is probably best avoided. Related to the question of standards is that of the units in which results should be expressed. In 1960 King and Campbell recommended the use of pmoles/ml as standard units, expressing these units in concentrations per ml or per litre. The subject of units in clinical enzyme assays is really in a sorry state! For example, King and Campbell converted various units to the suggested standard system of pmoles/min/litre. For alkaline phosphatase, 1 King-Armstrong unit/ 100 ml is equivalent to 7.1 standard units and 1 Bodansky unit/lOQ ml to 5.35 standard units; for amylase, 1 Somogyi unit/lOO ml is equivalent to 2.06 standard units. We thus have different units for different enzymes, and for the same enzyme estimated in different ways. Radin (1967) discusses in a general article the use of standards in enzyme assays and strongly supports the recommendations of the International Union of Biochemistry, i.e., to use pmoles of substrate transformed/ min. It would appear that in clinical chemistry the problem is still not fully resolved. Given that it is preferable to express results in absolute units, what is necessary to convert an instrument reading on an automated analyzer to such units? The first requirement is to know the relationship between instrument reading and pmoles of substrate or product. Although some enzyme reactions are based on the formation of rather illdefined coloured complexes, it is generally possible to define the reaction in rigorous chemical terms, by the correct use of standards. SubjecI index P. 218

70

AUTOMATED ENZYME ASSAYS

Thus, although the precise chemical nature of the blue product formed after reduction of phosphomolybdate may not be known, the assay can be calibrated with known amounts of inorganic phosphate. The correct procedure is to calibrate the instrument with a known, stable and well characterized substance. In the case of spectrophotometric assays using standard flow cells of known optical path, published values of molar extinction coefficients may be used, although it is as well to ascertain that the instrument used does in fact give the published absorbance values. In the Technicon system it is difficult to calculate values from published molar extinction coefficients because of the difficulty in obtaining an accurate measure of the true path length of light in the tubular flow cell, which relies on internal reflectance to increase the effective light absorption. Even with more standard cells, however, the observed absorption will depend on the sharpness of the absorption band of the chromophore and the spectral resolution of the instrument. Thus, most instruments give similar values with phosphate standards using the reduction of phosphomolybdate as an assay because the coloured complex has a flat absorption spectrum in the blue region. However, measurements based on the CI band of cytochrome c at 550 nm require considerable accuracy in the wavelength calibration of the instrument and will vary with the width of the waveband of incident light. For these reasons, it is preferable to use the general factor relating readings to molarity which I call the molar conversion factor (MCF) ; this is defined as the concentration of solute in ,umoles/ml that corresponds to an instrument reading of 1.O. In spectrophotometric assays it is directly related to the molar extinction coefficient as discussed more fully in 0 8.2.1. However, it may be used for any assay in which response of the instrument can be related to concentration of a known substance. If the instrument gives a linear response, one simply calculates one MCF value and multiplies all readings by it to give the final concentration in pmoles/ml. This is done in the computer program in appendix 1V. If there is no linear or other predictable relationship between reading and concentration, a series of MCF values must be obtained over the range of assay, and the appropriate

SINGLE-ENZYME ANALYSIS

71

value selected for a given reading by graphical methods or linear interpolation, preferably by computer. The calibration curve may be carried out by preparing a series of standard solutions manually and placing them in the sensor. However, if the instrument is fully calibrated (see Q 8.2.3) in terms of the flow rates in the various lines, the calibration curve may be performed automatically, using a gradientmaking system. In the actual enzyme assay, it is important to establish that the changes in instrument reading are due to changes in the concentration of the substance being measured and not to other effects. For example, if a mitochondria1 enzyme is being assayed, swelling of the mitochondria caused by addition of substrate may give a fall in absorbance that is unrelated to the transformation of that substrate. For this reason it is important to use correct blanks and to measure instrument zeros before and after the assay.

5.2. Determination of incubation time A more difficult problem than the calculation of MCF is the determination of the precise time of incubation. This does not arise in semiautomated methods or in interrupted-flow systems, since the progress curve is obtained automatically, and the only conversion necessary is from chart divisions to minutes or seconds. In the continuous-flow analyzer and probably in the discrete-sample analyzer certain problems occur. These are best illustrated by reference to the simple assay in Q 3.2. The enzyme is pumped through the enzyme line and meets the stream of reaction mixture on entering the jacketed mixing coil MC, in fig. 3.1. The reaction starts as soon as the enzyme and reaction mixture streams mix. Also, since in the flow system shown the reaction is not stopped at any particular time by a precipitant or filter, it will continue while the reaction mixture flows from the heating bath, where the bulk of the reaction occurs, to the colorimeter. We can measure the time taken for the reaction mixture to reach each point by using a short pulse of a suitable marker (e.g. strong ferricyanide solution for 2 sec). The time of passage through the jacketed mixing coil is 55 sec, through the two coils of the heating bath is 11 min 2 sec and from the Subjecl index P. 218

12

AUTOMATED ENZYME ASSAYS

heating bath to the colorimeter is only 43 sec. Since this last value is relatively small, we can assume that there was negligible cooling of the reaction mixture in this time and we can add the time to the total time in the heating bath. The time of incubation in the mixing coil before the heating bath is very difficult to evaluate. This is because part of the measured time in this coil is spent in (a) raising the temperature of the enzyme to that of the jacketed coil, (b) mixing the enzyme and reaction mixture, and (c) increasing the enzyme concentration until it reaches steady state. By dipping the enzyme line into a standard solution of marker, we can measure the ‘enzyme input’ curve precisely, i.e., determine how the

Y

0

t

I

I

1

2

Dye enters r e a c t i o n vessel

I

I

3 4 Time (min)

I

5

I

6

t

Dye e n t e r s sensor

Fig. 5.1. Error in estimate of incubation time caused by approach of enzyme concentration to steady state. A flow system with 6 min retention time in the incubation vessel was calibrated by placing dye in the enzyme line. The plot of effective enzyme concentration against time is given. The system has a half-wash time of 15 sec (see ch. 8). The total activity is proportional to the product of enzyme concentration and time, i.e., the area under the curve. If one ignores the hatched area, i.e., if one assumes that the enzyme concentration reached steady state instantaneously, the error is 5.7%. If one deducts two half-wash times (30 sec) from the retention time, the error is only I .7 %. (A system virtually reaches steady state in four half-wash times - see 8 8.2.7.)

SINGLE-ENZYME ANALYSIS

13

enzyme concentration rises in the coil. In an example of this (fig. 5.1) it can be seen that the enzyme has virtually reached its steady-state concentration in 90 sec. Assuming that the input curve is a straight line we could deduct half this time from the incubation time. It is difficult to measure the time for temperature equilibration in the coil. However, if the enzyme contributes only a small percent to the total volume of the reaction mixture, the effect is probably not great. With some effort, these factors could be measured precisely. The jacketed mixing coil as a whole only contributes a maximum of 7% to the rota1 incubation time, so if we arbitrarily assume that the effective incubation time in this coil is 50% of the measured delay time in it, our total error would not be very great. We may measure retention times by pumping markers through the systems and following their progress with a stop watch. A useful way of doing this is to introduce a marker into one of the lines for a very short time and to place a shutter between the light source and the flow cell momentarily as the marker passes the point being measured. This causes a vertical mark on the trace of the pen recorder, and when the marker ultimately reaches the colorirneter the sudden rise in absorbance clearly marks its entry into the flow cell. The distance between this mark and that caused by the shutter is measured, and the retention time calculated from the chart speed (see 5 9.3.3, and appendix IV, pp. 196,202). Another method is to determine the volume of the various parts of the system by filling them with fluid and pumping the contents into a measuring cyclinder. From the rate of pumping of fluid and gas we could then calculate retention times. Since the gas will expand in the heating bath the retention time would be affected by the temperature of the bath. The observed effect, however, is very small (fig. 5.2). The volume of the flow cell in the Technicon autoanalyzer (and indeed most well-designed autosensors) is small relative to the total volume of fluid-flowing, and retention time in it may be ignored for most purposes. However, this is not so with very slow flow rates (e.g. 1 ml/min or less). To conclude, with ‘average’ systems pumping 3-6 ml fluid per min and 1-2 coils in the heating bath, errors due to smearing of the enSubiecl index P. 218

74

AUTOMATED ENZYME ASSAYS

0 N c

m

% m

60-

>

+ 0

s

40-

.-

.I-

I

40

,

50

I

60

Bath temperature

70

("C)

Fig. 5.2. Effect of bath temperature on retention time. The manifold consisted simply of a 2 ml/min fluid line pumping water and an 0.8 ml/min air line. The observed effect (solid circles) is greater than would be expected from simple expansion of the gas (open circles), possibly because of change in peripheral resistance caused by a fall in viscosity. Note that a change of 1 "C in bath temperature in the range 30-40 "C gives only a 0.13 % change in retention time. (See Barrera et al. (1969) for a similar curve.)

zyme front, mixing in pre-heating bath coils and flow through the sensor are probably less than 5%. They are not strictly errors, but rather reproducible and systematic delays in the flow system which result in an effective loss in incubation time. The position is that although the effective incubation time may not be known to an accuracy of greater than 5y0,its reproducibility is far greater than this. This situation is satisfactory for most circumstances. However, if a precise estimate of the incubation time is required the following procedure is necessary. The assay is first carried out manually or semi-automatically. If possible, the enzyme plus reaction mixture should be introduced into the sensor of the actual autoanalyzer so that the progress curve can be determined with the same instrument and reagents. The

SI NGLE-ENZY ME ANALYSIS

75

period of time over which the assay is linear is determined and the assay is then repeated on the autoanalyzer. From the plot of reading against time on the manual or semi-automatic method, the time corresponding to the reading obtained with the autoanalyzer may be calculated. Once this has been done with one enzyme with a given flow system, the value for incubation time so obtained may be used for all other assays. Another method is to perform the assay with no coils other than the pre-heating bath coil and to substract the value so obtained from the value obtained with the complete system. In this way the unknown effective time of incubation in the mixing coil is eliminated from the calculations. A progress curve obtained by running the same assay with 0, 1,2,3, and 4 coils (fig. 5.3) illustrates the method of correcting for any incubation that occurs before the heating bath.

Incubation time (min)

Fig. 5.3. Measurement of progress curve by varying number of heating bath coils. incubationwith no heatingbath(mixingcoi1only), 1,2, 3, and 4 coils in two heating baths in series. L(+)-lactate dehydrogenase of yeast measured by reduction of potassium ferricyanide.(From Roodyn 1969.)

5.3. Measurement of the progress curve This leads to the difficult problem of determining the full progress curve in a continuous-flow analyzer with a reasonably simple and Subject index P. 218

76

AUTOMATED ENZYME ASSAYS

convenient flow system. One solution, which is rather expensive, is to use a multi-channel analyzer with different times of incubation in each channel. A simple dual-channel analyzer for this purpose is shown in fig. 5.4 and really consists of two manifolds of the basic design in fig. 3.1. Another design is to have one manifold with two colorimeters in series (fig. 5.5). By using a previously determined zero-time reaction we can obtain a three-point progress curve with such systems. (Note, however, that a sigmoid curve may also fit a three-point curve.) A 'family' of three-point progress curves is shown in fig. 5.6 where lactate dehydrogenase was studied at various temperatures. The method revealed that the assay was only linear below 40 "C,because of inactivation in the heating coils at the higher temperatures. Other workers have used dual-channel autoanalyzers for similar reasons. Schuel et al.

Fig. 5.4. Dual-channel multi-enzyme analyzer. The lower channel has one coil in the heating bath, the upper two. As a result the time of incubation in the lower channel is approximately half that in the upper. B: buffer; CI, CZ:colorimeters; D: de-bubbler; HB: heating baths; MI, Mz: mixers; MC: mixing coils; PI,Pz: proportioning pumps; S: sampler; R1, Rz: recorders; W: waste. (From Roodyn 1967a.)

77

SINGLE-ENZYME ANALYSIS

COL 2

Fig. 5.5. Alternative-flow system to give two incubation times. After the reaction mixture has passed through the first heating bath (HB 1) the de-bubbled stream (D) passes through the first colorirneter (COL 1). The remainder of the gas-segmented stream passes through the next bath (HB 2) and colorirneter (COL 2). An enzyme gradient is generated by the two mixers (Mi, Mz) and a pump. G R : gradient tube; JMC: jacketed mixing coil; MC: mixing coil; S: sampler; W: waste; W1, W2: waste lines. (From Roodyn 1969.)

Temperature of assay ("C)

3 * Lu

03-

2

r 1.

._

Y 02-

!i

c

w

-

0

8 Incubation time (min)

12

Fig. 5.6. Examples of three-point progress curves obtained with a dual-channel analyzer. L(+)-lactate dehydrogenase assayed at different temperatures. Note that the reaction was not linear at 40 and 45 "C.(From Roodyn 1969.) Subject index P. 218

78

AUTOMATED ENZYME ASSAYS

-

Pump 1

su B

1

G -co -E

Fig. 5.7. Four-channel enzyme analyzer. The enzyme, buffer, co-factor and substrate streams are all split into 4 channels with 0, &, 1 and 1& coils, respectively. The system requires 2 standard proportioning pumps, two heating baths, and a method of measuring four absorbance values (i.e., four colorirneters or a 4-cell continuous-flow sample changer in a spectrophotometer). The system is continuous flow and will give four-point progress curves (or five-point curves if one includes previously determined zero-time values). B : buffer; c1-4: colorimeters; CO: cofactor; E: enzyme; G: gas; HB: heating bath with size of &il; JMC: jacketed mixing coil; MC: mixing coil; SU:substrate; W: waste.

SINGLE-ENZYME AhALYSIS

79

(1964) used a dual-channel system for assay of cytochrome oxidase, with short incubation times, 20 sec in one channel and 120 sec in the other. Pitot et al. (1965) used a two-channel system in which the delay times in each channel were chosen from the linear region of the progress curve that had been determined previously by manual methods. The two streams passed through a dual colorimeter and the difference in reading between them was taken as a measure of the rate. More complex systems are possible using more sensors; fig. 5.7 shows a fourchannel system, combining the designs in figs. 5.4 and 5.5. Each channel has to be calibrated separately in terms of line dilution factors and molar conversion factors. Without computer processing the calculation of results in such systems can become burdensome. An alternative system that resembles the action of a discrete-sample analyzer is shown in fig. 5.8. The entire reaction mixture is placed in the sample cup and by using two 3.9 ml/min lines connected in parallel, the entire contents of the cup are pumped into the reaction vessel in

Fig. 5.8. Alternative method of operating the autoanalyzer for enzyme assays. Cups containing 1.6 ml of complete reaction mixture are loaded in the sampler, (Sl), and emptied by two wide pump tubes into a jacketed reaction vessel (R), stirred by a magnetic stirrer (M). At the same time another sampler (SZ) working in synchrony with SIis used to load 0.4 ml enzyme sample into the reaction mixture. The cups in S1 and SZ are emptied after 0.2 min pumping and no more fluid is pumped into R. The contents of R are emptied continuously through the flow cell (FC) of the colorimeter (COL) at a rate that gives a continuous reading for over 3 min. At the end of this time, R becomes empty and the next aliquots of enzyme and reaction mixture are pumped in from the samplers. (The number on each line indicates the flow rate in ml/rnin). Subjecl index p. 218

80

AUTOMATED ENZYME ASSAYS

0.2 min. At the same time a small volume of enzyme is pumped into the vessel from a line controlled by the same sampler crook. The reaction vessel is emptied continuously through the flow cell at a rate which allows 3 min for incubation before the vessel runs dry. After another minute, the next sample and reaction mixture are pumped in. In this way, a continuous family of progress curves may be obtained without actually stopping the flow of the instrument, as in the interrupted-flow methods. However the method suffers from the same disadvantage of being a sequential assay which must have short incubation times for a reasonable overall rate of sampling. Several authors have described the use of the sampler I1 module of the Technicon system as the reaction vessel and the progress curve is followed as the cup is emptied. For example, Berry and Walli (1966) measured lactate dehydrogenase in serum by this method and determined the reaction rate from the sample peak, by selecting a region corresponding to steady-state enzyme concentration. A similar method has been employed by Brown and Ebner (1967).

5.4. Relation between enzyme activity and protein concentration A second important criterion for valid enzyme assay, perhaps more important than measurement of the progress curve, is that the rate should be measured under conditions in which it is proportional to enzyme concentration. In fact, although preferable, it is not essential to have linear progress curves in order to have proportionality to enzyme concentration. Thus, in fig. 5.9 some hypothetical reactions that are far from linear with respect to time are still proportional to enzyme concentration. Nevertheless, such proportionality is not always easy to achieve and, for technical reasons, is usually checked less often than the progress curve. In manual assays this check is usually carried out by repeating the assay at 2 or 3 different protein concentrations for each sample. Sometimes, authors establish the range of linearity at the beginning of a series of experiments and assume that all subsequent values with a single protein concentration that fall

81

SINGLE-EN,ZYME ANALYSIS

in this range are linear. With an automated assay system one can perform such calibration readily by placing the enzyme in a linear gradient-making system. In fact, it is easier to check proportionality to enzyme concentration on a routine basis than to follow the progress curve. (The use of gradient-making systems in this respect is discussed more fully in $9 6.3, 7.5, and 8.2.5; see also Roodyn and Maroudas (1968).)

5.5. Enzyme monitoring Having discussed some of the problems and principles involved in S.E.A. I would like to discuss its use in ‘enzyme monitoring’. At the


E0

1

2 Time (min)

,

,

3

4 4 min 3 rnin 2 rnin

1 rnin

0

1

2

4

Protein concentration ( a r b i t r a r y units)

Fig. 5.9. Possible linear relation between enzyme activity and protein concentration with non-linear progress curves. Four hypothetical protein concentrations (PI-4) give non-linear progress curves (a), but all activities me linear with respect to protein concentration (b). Subject index P. 218

82

AUTOMATED ENZYME ASSAYS

moment the most convenient systems for measuring activity in a continuous flow of enzyme are the continuous-flow systems, although interrupted-flow systems have been used for this purpose. Discretesample analyzers are not so suited for monitoring, although presumably they could be connected to the enzyme stream by a suitable fraction collector. An important example of monitoring is the study of the elution of enzymes from columns. For example, Mundry (1965) described a continuous-flow system for following the purification of ribonuclease. Column effluentsweer analyzed for glucose-6-phosphate dehydrogenase, lactate dehydrogenase and phosphoglucomutase by Hoober and Bernstein (1964), and by Catravas and Lash (1966) for serum enzymes. It is useful in such methods to have a simultaneous measure of the protein content of the stream, so that immediate calculations may be made of the degree of purification achieved. Some workers have described double-flow systems for the simultaneous estimation of enzyme and protein; an example (fig. 5.10) from a paper by Scheuerbrandt (1965) is included to illustrate the complexity of the flow systems now used. (The reader may have noticed a resemblance between these diagrams and simple electronic circuits. The analogy may be important. Just as electronic systemsof extraordinary complexity evolved from simple components that could be arranged in a very large number of ways, so future flow systems may well reach similar levels of complexity.) Enzyme monitoring has been used to follow the level of enzymes in animal or human blood which flows directly into the analyzer from a special venous cannula. For example, Serrone et al. (1965) monitored whole-blood cholinesterase in monkeys and studied the effects of various pharmaceutical agents. Groff et al. (1966) used a three-channel autoanalyzer for monitoring blood cholinesterases. By having different substrates in each channel they were able to distinguish effects on plasma and erythrocyte enzymes. Unfortunately, because of difficulties in cell breakage, it is not easy to monitor levels of intracellular enzymes during the growth of microbial cultures. Avanzini et al. (1967),however,have followed respiratory enzymesin bacterial cultures by using tetrazolium red as an electron acceptor.

Automatic determination of a decarboxylase flow diagram

Water CO, -free

1

Bf -_

KOH Wash b o t t l e

Fig. 5.10. Simultaneous measurement of enzyme activity and protein concentra-

tion. (From Scheuerbrandt (1969,by courtesy of the author.)

84

AUTOMATED ENZYME ASSAYS

The monitoring of enzyme patterns in cell particles in various forms of gradient centrifuging promises to be a most productive technique and Leighton et al. (1968) have recently used the autoanalyzer extensively to characterize rat-liver sub-cellular fractions. We have recently monitored sucrose gradients of cell particles from yeast, assaying for a number of enzymes. A dual channel analyzer was connected to the sucrose gradient tube. One channel measured enzyme activity and the other was the blank (fig. 5.1 1). There was thus a continuous correction of the blank during analysis of the gradient. The stream from the gradient tube was also split to provide samples for chemical and radioactivity analyses, which were in fact performed manually but could well have been carried out automatically. Typical sedimentationcurves obtained by t h i s system are shown in fig. 5.12. The zonal ultracentrifuge developed by N. G. Anderson and his colleagues is a most powerful instrument for centrifugal analysis and fractionation, and in theory at any rate, is well suited for on-line enzyme monitoring. Schuel and Anderson (1964) assayed the effluent from the centrifuge for acid Pump1 1 -Substrate

Col2

Gradient tube

Fig. 5.11. Two-channel system for analyzing sucrose gradients. The upper channel has the full reaction mixture and the lower channel measures the rate in the absence of substrate. (Symbols as in fig. 5.5.) (From Roodyn and Grivell 1969.)

85

SINGLE-ENZYME ANALYSIS

phosphatase during a fractionation of rat-liver particles (fig. 5.13). Schuel et al. (1964) performed similar experiments with cytochrome oxidase. The analysis of effluent from the zonal ultracentrifuge is not without difficulties.With present rotors, the volume of fluid pumped out during the emptying of the rotor is too great for most analyzers, necessitating some system for stream splitting. Also the enzyme concentration may be low if the analysis has been carried out with quantities of material that give the best sedimentation patterns. 'The chief difficulty is the high sucrose concentration in the effluent which may interfere with the

0.3--

2k 5?

0.4

IP-J

$j 0.2-a c

e

0

Q

0,

3 0.1 U

m 0 .P

L2

0-

I

0

t

Bdttorn

10

I

ml

20

t

Top

Fig. 5.12. Combined chemical and enzymic analysis of sucrose gradients. Radioactive yeast mitochondria were centrifuged in a sucrose gradient and the stream from the gradient tube split into one half for enzyme analysis and the other for determination of protein (PR)and radioactivity (CPM). (From Roodyn and Grivelll969.) Subject index P. 218

98

SAVSSV ~ W A Z N Bamvwouv

86

AUTOMATED ENZYME ASSAYS

9t OP EE +MI asoJ>nS

(elo

99

s9 09EE

09

OE

EZ OZOL

0

0s ot OE-

t, 3

g ozN

:ElP)

U 0

g

5

O'L-

2'0 0091

OOPL OOZL

0001

008

009

OOP

002

0

Fig. 5.13. Enzyme monitoring of effluent from zonal ultracentrifuge. Acid phenyl phosphatase measured atpH 4.1,4.8,5.4 and 5.9 in effluent from zonal ultracentrifuge after centrifuging of rat-liver homogenate. (From Schuel and Anderson (1964), by courtesy of the authors.)

enzyme assay and also prevent mixing of reagents in the lines of the analyzer. However, these problems are not fundamental and there is little doubt that the zonal ultracentrifuge monitored by suitable automated enzyme analyzers will be a useful tool in cell fractionation. There is thus little doubt that the application of automated S.E.A. methods to enzyme sampling and enzyme monitoring will have a great impact on biochemistry. The use of these techniques in enzyme characterization is discussed in ch. 7.

CHAPTER 6

Multiple-enzyme analysis (M.E. A.)

6.1. Principles of multiple-enzyme analysis (M.E.A.) It is often necessary to assay more than one enzyme. Of course this could be achieved by simply increasing the number of channels or analyzers. For example Passen and Gennaro (1966) described a dualchannel analyzer for the simultaneous measurement of glutamateoxaloacetate transaminase and lactate dehydrogenase in serum. However, a much simpler and cheaper method for multiple analysis was suggested by the author in 1964. The usual procedure in automated assays in the Technicon system is to place the different enzymes on the sample plate and aspirate them into a continuous stream of substrate plus buffer (‘enzyme sampling’). However, if a series of substrates is placed in the sampler module and a mixture of enzymes, for example a tissue homogenate, is pumped through the enzyme line, as each substrate is sampled, the appropriate enzyme in the mixture is analyzed. The principle of the multiple-enzyme analysis system (Roodyn 1964, 1965b) is shown in fig. 6.la, and the type of trace obtained is shown in fig. 6.lb. In its simplest form, multiple-enzyme analysis (M.E.A.) is suited to enzymes that have many common features in their assay requirements, For example any of the groups listed in table 6.1 could be adapted to the system by the methods shown. The usual requirement is a common buffer and a common sensor, although the actual product need not be the same. Thus both NAD- and NADP-linked reactions could be followed in the same sensor at 340 nm, a continuous-flow 87

subiecr index p . 218

88

AUTOMATED ENZYME ASSAYS

SUbstrates

MC MC y / / / / / M/I//?

I

Common buffer, co-factors Gas Enzyme mixture

WL

f

2

M

._

-j

1

-

1 Remove enzymes Time (min)

Fig. 6.1. Principles of multiple-enzyme analysis. (a) A series of substrates are placed in the sampler S. These are mixed sequentially with a buffer common t o all enzyme assays and then with the enzyme mixture. (b) An idealized trace. The activity of the enzyme attacking the appropriate substrate is indicated by a peak. (From Roodyn 1969.)

pH meter could monitor any reaction that caused a change in pH, and so on. All the assays are most conveniently carried out at the same pH. However, if this is not possible apH gradient-makingsystem could

89

MULTIPLE-ENZYME ANALYSIS

TABLE 6.1. Examples of groups of enzymes readily amenable to multiple-enzyme analysis. Group

Common factor in assay

Arnidases

Release of ammonia

Aminotransferases

Formation or disappearance of glutamate or a-oxoglutarate

Decarboxylases

Release of COZ

Dehydrogenases (general)

Variety of natural and artificial electron acceptors.

Esterases

Change in pH or release of chromogenic alcohol

~~

Ferricyanide reductases

Fall in absorption at 420 nm

Glycosidases

Appearance of reducing sugar. For glucosidases appearance of glucose.

NAD-, NADP-linked dehydrogenases, Fluorescence measurement or change in or assays linked with these enzymes absorption at 340 nm Phosphatases

Release of inorganic phosphate

Phosphotransferases

Formation or disappearance of ATP

Sulphatases

Release of sulphate

be incorporated. Some examples of analyses performed in our laboratory by this method are given in fig. 6.2, and in Roodyn (1964, 1965c, 1967a). The Clark oxygen electrode was converted for continuous flow and used in a flow system (fig. 6.3) for M.E.A. of oxidases. Although it is convenient to have some common features in the reaction mixtures for the various forms of assay, in fact it is possible to use completely unrelated reaction mixtures prepared in concentrated form and simply diluted with water. The only requirement here is that a common sensor is used. Such a system requires a very simple manifold (fig. 6.4), although it does require the previous preparation of the various reaction mixtures. Preliminary experiments with this system have indicated that it has great potentiality. Subirc! index

I).

218

90

AUTOMATED ENZYME ASSAYS

Substrate AMP

ADP ATP Glucose-I- P Glucose-6-P

-

L

3 P-GI ycera te

Fructose-dl

-P

Fig. 6.2. Study of phosphatases by multiple-enzyme analysis. Yeast homogenate was incubated with a range of phosphate esters in presence or absence of Mg++ or Cat+ ions. Inorganic P liberated was measured by method of Fiske and Subbarow (1925), adapted to the autoanalyzer. Dialyzer was used to separate inorganic P from protein. (From Roodyn 1965c.)

n Stirrer

Fig. 6.3. Flow system for multiple-enzyme analysis of oxidases. Numbers refer to flow rate in rnl/min. (From Roodyn 1964.)

91

MULTIPLE-ENZYME ANALYSIS

~,

n

JMC

,

7

G

a

s

p

Enzyme mixture

Colorirneter Recorder

Fig. 6.4. Multiple-enzyme analysis with sampling of complete reaction mixture. The various reaction mixtures are prepared previously and loaded into the sampler (S) which should be the sampler I1 module. D: de-bubbler; FC: flow cell; HB: heating bath; JMC: jacketed mixing coil; W: waste.

TABLE 6.2 Ferricyanide reductases in yeast strains studied by multiple-enzyme analysis. Strain

Ferricyanide reductase (,u moles/min/mg protein

Normal strains 4CI 10 21 40 45 188 XI 40 X 41 ‘Petite strains’ 45 sp 45 ac 4CI sp 4C1 sp/Co 188 uv 45 ac x 188 uv 45 sp x 4 c1 sp

N)

NADH-

L(+)-lactate

D(-)-lactate

Succinate

2.64 4.81 4.20 0.95 2.96 1.80 2.90 3.92

2.09 0.97 0.60 0.17 1.40 0.35

1.78 1.74

0.02 0 0 0.33 0.10 0.38 0.01 0.01

0.01 0.10 0.06 0.01 0.15 0.01 0.06 0.15

2.42 2.03 1.30 1.42 0.94 1.57 1 .oo

0.15 0.03 0.09 0.01 0 0.04 0

0.80 0.73 0.13 0.39 0.60 0.52 0.22

0.02 0 0 0 0 0 0 Subiec: index P. 218

92

AUTOMATED ENZYME ASSAYS

6.2. Enzyme patterns The most obvious use of M.E.A. is as a semi-quantitative system to ‘scan’ a given preparation for groups of enzymes. There are many situations in which such a survey is of use and as an example I will consider some results of the study of ferricyanide reductases in yeast mutants (table 6.2) (Roodyn gnd Wilkie 1967). This approach could be of use in clinical medicine in attempts to correlate changes in enzyme patterns with pathological change. Measurement of enzyme patterns raises certain problems in the presentation and assessment of results. For example, the results of the survey in table 6.3 are presented in pmoles substrate/min/mg protein N. However, the values of interest are not only the absolute activities but also two relative sets of values. The first of these is the relationship between the enzyme activities in the parent, and the mutant strains. The second set is concerned with the pattern of enzymes in any one strain. If we consider the ratios of parent strain to mutant strain first, we can see that there is a characteristic change in each case for all four enzymes TABLE 6.3 Analysis of data in table 6.2.: comparison of mutant with parent strains. Respiratory deficient mutant strain

Enzyme activity in mutant Enzyme activity in parent strain* NADH L(+)-lactate D( -)-lactate Succinate dehydrogenase dehydrogenase dehydrogenase dehydrogenase

45 sp 45 ac 4c1 sp

4c1 sp/co 188 uv 45 ac x 188 uv 45 sp x 4C1 sp

0.82 (F) 0.41 (F) 0.49 (F) 0.54 (F) 0.52 (F) 0.53 (F) 0.33 (F)

0.11 (F) 0.02 (F) 0.04 (F) 0.005 (F) 0 (F) 0.03 (F) 0 (F)

8.0 (R) 7.3 (R)

6.5 (R) 19.5 (R) 1.6 (R) 5.2 (R) 2.2 (R)

0.13 (F) 0 (F) 0 (F) 0 0 0 0

(F) (F) (F) (F)

* F: fall in activity relative to parent strain, R : rise in activity. Note that although the quantitative values are different all mutants show the pattern F,F,R,F.

93

MULTIPLE-ENZYME ANALYSIS

(table 6.3). Similarly, if we sum all the activities in a given strain and express each activity as a fraction of this sum, we can obtain a series of patterns (fig. 6.5) which show characteristic shapes for parent and mutant strains. Thus, analysis of groups of enzymes yields data that can be tabulated and plotted in many ways, and it is not always clear how best to demonstrate the effects or changes under study. Pette et al. (1962) have made similar multi-enzyme studies on mitochondria, using manual methods of assay, however. Nevertheless, they have made interesting correlation analyses within the groups, and have demonstrated the existence of constant-proportion groups of enzymes. Since we are only at the beginning of experiments with groups of >,

.c _

>

‘Z

10-

(a)

m aJ

5

1-

C

(b)

(4‘21 sp/Co) (188 uv) (45spx4C1 sp) 44

U

m

E, ti

10-

N v

1-

X

0

Fig. 6.5. Enzyme patterns in yeast. N: NADH dehydrogenase; L: L(+)-lactate dehydrogenase; D : D( -)-lactate dehydrogenase; S: succinate dehydrogenase. Data from table 6.3, strain number in brackets. (a) Typical wild-type pattern; (b) typical ‘petite’ pattern; (c) ‘intermediate’-typepattern. Subject index p . 218

94

AUTOMATED ENZYME ASSAYS

enzymes, it would be helpful to anticipate some of the problems in data handling that may arise from such studies. There is no doubt that the computer will be used to search for correlations in such data. As an example of this, we can refer to the above study on yeast mutants. It appears that the characteristic change during the appearance of respiratory deficient mutants is a partial fall in NADH dehydrogenase, a serious fall in I,(+)-lactate dehydrogenase and succinate dehydrogenase and a significant rise in D( -)-lactate dehydrogenase. The effect is thus characterized by the behaviour of 4 enzymes. The changes in enzyme pattern during the glucose repression-derepression cycle of yeast (fig. 6.6), show an identical effect at the period of full repression (9 hr). Thus, although the absolute enzyme activities may not be identical in the two sets of experiments, the overall change in 300

I

0

1

Days of growth

I

I

2

3

Fig. 6.6. Variation in enzyme pattern during growth of yeast. Yeast grown on glucose medium. N: NADH dehydrogenase; L: L(f)-lactate dehydrogenase; D: D(-)-lactate dehydrogenase; S : succinate dehydrogenase. (From Roodyn 1969.)

95

MULTIPLE-ENZYME ANALYSIS

pattern is strikingly similar, indicating that a similar process is occurring during both glucose repression and induction of the mutant. The possible correlation of changes in enzyme pattern with genetic alterations by such techniques could well be a useful line for future investigation.

7 0 3 ’ 2

3

Analyzer

Enzyme+ Protein

Fig. 6.7.Multiple-enzyme analysis withenzyme gradient and recycling of substrates. A group of three substrates is cycled four times. Stock enzyme is pumped into a gradient-making system to give a linear increase in protein concentration during the assay. The upper diagram shows the general arrangement of the system, and the lower diagram shows how the protein concentration varies during the assay, and how four values are obtained for each of the enzymes (hypothetical results). Subject index p . 218

96

AUTOMATED ENZYME ASSAYS

6.3. Enzyme gradients One of the difficulties of M.E.A. is that enzymes do not occur naturally in equal concentrations. Thus, a crude tissue homogenate may contain 10,100 or even loo0 times more of one enzyme than another. It is thus difficult to assay all enzymes at a suitable protein concentration. This difficulty may be overcome by using an ‘enzyme gradient’, and performing some assays at low and some at high protein concentration. In order to obtain several values for each enzyme, the substrates may be placed in repeating groups on the sample table. The principle of such a system is shown in fig. 6.7 and some typical results are in fig. 6.8. The actual trace obtained during an analysis by multiple-enzyme analysis with an enzyme gradient is quite complex, and an example is given in Roodyn (1967b). It may be seen from these traces that not all -08

u

-01

Cup number

Fig. 6.8. Typical result with system described in fig. 6.7. Three ferricyanide reductases in a yeast homogenate were measured. Enzyme activities and protein concentrations were plotted and calculated by computer (see ch. 9 and appendix IV) L: L(+)-lactate dehydrogenase; D: D( -)-lactate dehydrogenase; S: succinate dehydrogenase; PR: protein. The sampler contained repetitive cycles of L(+)lactate, D( -)-lactate and succinate and the reaction was followed by measuring the reduction of potassium ferricyanide at 420 nm.

MULTIPLE-ENZYME ANALYSIS

91

the enzymes showed perfect linearity over the entire range of protein concentrations tested. In particular NADH dehydrogenase gave linear values only at the lowest protein concentrations. By the use of suitable gradient-making systems and substrate patterns on the sampler table it is thus possible to have conditions that give valid assays for all the enzymes in the group. The experiment in fig. 6.8 was in fact carried out on a dual-channel analyzer after determination of the zero-time reading, so that at the same time a primitive three-point progress curve was obtained for each assay. Beck and Tappel (1967), and Tappel and Beck (1967) describe a multiple-enzyme monitoring system for studying 5 lysosomal enzymes (fig. 6.9). By using two samplers, one for the substrates and one for enzyme samples, the entire process was automated. (In the systems that I have described above, the enzyme preparation has to be changed manually between assays.) Strandjord and Clayson (1966a) have used a sampler with two concentric circles of holes. The substrates were

Flow r a t e

C o l o r i r n e t e r Recorder

Colorimeter

Fig. 6.9. Multiple-enzyme analysis with enzyme sampling.Two samplers were used, one with enzyme samples, and the other with a range of substrates to study 6 lysosomal enzymes. The sequence of sampling was controlled by timer relays. (Numbers refer to flow rates in ml/min.) (From Tappel and Beck (1967), by courtesy of the authors.) Subjecf index P. 218

98

AUTOMATED ENZYME ASSAYS

placed in the inner circle and serum enzymes in the outer, and several dehydrogenases were studied at the same time.

6.4. Multi-channel analyzers As mentioned in Q 6.1, one can obtain multiple analysis merely by increasing the number of analytical channels. The simplest case of the dual-channel multiple-enzyme analyzer has been mentioned above in connection with obtaining a progress curve. The dual-channel system could, of course, be used for many other purposes. As an example,

”

IV

LU

4-

Cup no.(proportional to protein concentrat ion) Fig. 6.10. Simultaneous study of enzymes in normal and mutant yeast using a dual-channel multi-enzyme analyzer. (a) ‘petite’ strain; (b) normal strain. Normal yeast, and a UV-induced petite strain were studied simultaneouslyusing an enzymegradient system. Enzyme activity was followed by measuring reduction of potassium ferricyanideat 420 nm by NADH, NADPH,t(+)-lactate (L),and succinate (S). The protein concentration increased linearly during the experiment and was similar in both channels. Note the disappearance of L(+)-lactate dehydrogenasein the ‘petite’ and the change in the ratio of the NADH/NADPH dehydrogenases.

MULTIPLE-ENZYME ANALYSIS

99

simultaneous comparison of normal and mutant yeasts was obtained with a dual-channel analyzer with separate enzyme lines (fig. 6.10). Another example is a dual-channel analyzer for the simultaneous determination of ferricyanide reductases and NAD(P)-linked dehydrogenases that has been developed in our laboratory. Provided the money is available, there is no limit to the complexity of such systems! One channel may be used for the normal assay, and the other to study the effect of inhibitors. For example, Green et al. (1966) used a dualchannel system to differentiate serum phosphatase isoenzymes by having specific inhibitors in one channel only. A three-channel system for studying cholinesterases has been described by Groff et al. (1966), each channel containing a different substrate. The obvious extension of such an approach is to construct large multi-channel systems. Perhaps the most striking of these is the AGA system which is a complex analytical centre, rather than an automatic analyzer. Specifically designed for clinical work, it measures many components in body fluids including several enzymes. It is described more fully in appendix I11 (p. 179). A similar but somewhat less complex system is the Vickers Multichannel 300 apparatus which is a discrete-sample analyzer in which up to 12 channels can operate simultaneously. This again is described more fully in appendix 111. The Technicon SMA/l2 system is a continuous-flow multi-channel analyzer that makes 12 different measurements on the sample, including the enzymes alkaline phosphatase, lactate dehydrogenase and serum glutamate-oxaloacetate transaminase (SGOT). The general arrangement is shown in fig. 6.11. and the output can either be used directly for computer processing (see 6 8.1) or is convenientlyrepresented on simple charts. The sequence in which the 12 analytical channels are read is carefully controlled so that a single record may be made from many simultaneous tests. The principles of this timing method are shown in fig. 6.12. It is not within the scope of this book to give a detailed account of these multi-channel systems and the reader is referred to Skeggs and Hochstrasser (1964) for a description of the earlier 8-channel Technicon system which exemplifies this type of approach. Subject index P , 218

100

AUTOMATED ENZYME ASSAYS

Substrate Air

Cresol ph thalei n boa0

Fig. 6.11. General arrangements for sequential multiple analysis. 12 different components may be measured by this system. The figure shows simplified flow diagrams for analysis (from top to bottom) of alkaline phosphatase, glutamateoxalacetate transaminase, total bilirubin, total protein, albumin, COa, sodium, potassium, chloride, blood urea nitrogen, glucose, and calcium. The samples are loaded on the sample plate (SP), diluted, dialysed and distributed lo the various analytical channels. (From Whitehead (1965), by courtesy of the author.)

101

MULTIPLE-ENZYME ANALYSIS

Fig. 6.12. Method for making a single record for 12 simultaneous tests. The total assay in each case is depicted by dotted lines and the time during which recording takes place by bold lines. The final trace is given below. (From Whitehead (1965), by courtesy of the author.)

It is clear that all the problems at present studied by S.E.A. methods could be extended by using multiple analysis. However, apart from the clinical multi-channel analyzers, multiple-enzyme analysis is still rarely used and its full potentialities have yet to be exploited.

Subiecr index p. 218

CHAPTER 7

Enzyme characterization

Schwartz and Bodansky (1968) have recently reviewed automated methods for studying enzyme kinetics. In this chapter I will discuss the simpler kinetic measurements that are made in order to characterize enzymes, either for more detailed analysis of their mode of action, or more usually, in order to develop satisfactory assay systems. These measurements usually entail determination of substrate specificity, Michaelis constants, p H and temperature optima, thermal stability, tests for requirements for co-factors, and effect of ions. All these studies lend themselves admirably to automation. Tappel and Beck (1965) carried out some pioneering experiments in this field and demonstrated the great potentialities of the continuous-flow analyzer in kinetic studies. However, automation in the full sense has not yet had much influence on such studies; in particular, it is still comparatively rare to see Michaelis constants determined with automatic substrategradient devices as described below.

7.1. Substrate speciJicity and Michaelis constants The determination of substrate specificityis simple, using a flow system similar to that used for M.E.A. Possible substrates are placed on the sampler table and aspirated into a continuous stream of enzyme. For example, Roodyn (196%) used an M.E.A. flow system for the determination of the chain-length specificity of a crude preparation of yeast alcohol dehydrogenase. The determination of Michaelis constant is 102

103

ENZYME CHARACTERIZATION

15 0.3

0

:10

w

0.2

4

c

0

m

U

0

L

.-Q

$ 5

m

C

Substrate concentration (mM) 3 6 ~

9 ~~

1 2 3 Reciprocal of substrate concentration (rnM-')

Fig. 7.1. Determination of Michaelis constant by varying substrate concentration in sample cups. A series of solutions of sodium malate of increasing concentration were placed in the sampler and aspirated into a continuous stream of enzyme. Malate dehydrogenase in a yeast homogenate was measured by following the reduction of NAD+ at 340 nm. e: Direct plot of change in E340 against substrate concentration. 0 : Plot of reciprocal of change in ES40 against reciprocal of substrate concentration. (From Roodyn 1965b.)

also relatively simple. A series of solutions of substrate may be placed on the sampler table as with yeast malate dehydrogenase (fig. 7.1). It can be seen that the classical linear double reciprocal plot is obtained (Lineweaver-Burke plot). Stein et al. (1965) performed similar experiments with ATPase. With multiple-enzyme analysis this may be repeated with every substrate that is attacked, so that the effect of substrate concentration on enzyme activities may be studied at the same time with a group of enzymes. An example of this is given in Roodyn (1967b), with 3 dehydrogenases in yeast. In order to avoid having to prepare several different dilutions of substrate solution, a gradient-making device may be used to prepare a Subjccl index P. 218

104

AUTOMATED ENZYME ASSAYS

'substrate gradient'. In this way one obtains an automatic plot of activity against substrate concentration. A very convenient way of preparing gradients in the autoanalyzer is to use the proportioning pump to pump fluid into and out of a mixing vessel. In this way, the gradient-making system is an integral part of the flow system. If the rate of emptying of the mixing vessel is exactly twice the rate of filling, a linear gradient results (Davis et al. 1965; Ayad et al. 1967) (see 8 8.2.5). Using this method, one can obtain a continuous trace of the sort shown in fig. 7.2 with yeast L(+)-lactate dehydrogenase. The

l

d

'

I

I

I

'

I I I I I I I I Chart divisions (substrate concentration)

'

1

Fig.7.2.Determinationof Michaelis constant by use of substrate gradient.Gradientmaking system attached to substrate line. L(+)-lactate (0.1 M) pumped into mixing vessel and linear gradient in lactate concentration established. L(+)-lactate dehydrogenase measured by fall in E4ao on reduction of potassium ferricyanide. The curve gave a linear Lineweaver-Burke plot. (From Roodyn 1969.)

Michaelis constant may be calculated from the half-maximal velocity or the data may be replotted as a Lineweaver-Burke plot (see legend), or an Eadie plot (Barrera et al. 1969). It is a relatively easy matter to perform such conversions on a computer and there is no doubt that such a system would have great value in determining kinetic constants with rapidity and precision. The same approach may be used to study the action of inhibitors. A rapid survey of possible inhibitors could be made by placing them on the sample table. Similarly, gradient-making

ENZYME CHARACTERIZATION

105

systems could be used to determine K,automatically. Schwartz and Bodansky (1966) used a gradient-making system to study the effect of activators and inhibitors on a number of enzymes. They used a nonlinear gradient with a constant-volume mixer as follows: Substrate or inhibitor was placed in a measuring cylinder. Water was pumped into the cylinder and the mixture was emptied at the same rate into the analyzer, so that the volume remained constant and the substrate or inhibitor concentration fell steadily in a non-linear way (see 0 8.2.5). The curves obtained on the analyzer thus gave the values with maximum concentrations of substrate or inhibitor at first and then showed the effect of their progressive dilution. Recently, Illingworth and Tipton (1969) have developed an automated apparatus for determining Michaelis curves. Power-driven syringes with an integral mixer produce a linear gradient of substrate which is combined with fixed proportions of enzyme. After passing through a reaction coil, the products are measured in a 4 cm flow cell in a specially modified spectrophotometer which can measure changes in NADH concentration in the region of 10-7 M. The full potentialities of more complex gradient systems have not yet been exploited. For example by using the ‘autograd’ system used in amino acid analysis (see ‘Techniques in Amino Acid Analysis’, 1966) it may be possible to expand certain regions of the classical Michaelis or Lineweaver-Burke plots so as to improve the precision of assay. For example, by lesseningthe gradient slope at higher substrate concentrations one may be able to obtain more precise values for the intercept of the latter plot with the l/v axis. The use of gradientmaking systems facilitates the multi-parameter analysis of enzymes. For example, Birkett et al. (1967) used a series of substrate gradients with different inhibitor concentrations in studying the kinetics of alkaline phosphatase (see also Posen et al. 1967). It is easy to imagine systems in which substrates, inhibitors, activators or co-factors are all rapidly varied so as to provide a multiplicity of assay conditions.

Subiecr Index P. 218

106

AUTOMATED ENZYME ASSAYS

7.2. p H optimum The automatic determination ofpH optimum poses certain difficulties. The easiest approach is to repeat the assay several times with a series of buffers at different pH. However, this is only semi-automatic, and pH profile of J3-glucuronidase activity rExtrapolated absorbance A

P-Nitrophenvl-R-D-

End pH=3.92

line

Fig. 7.3. Automatic determination of pH optimum. #I-glucuronidase measured. ApH gradient was established in the buffer line and monitored by a flowpH meter. (From Tappel and Beck (1965), by courtesy of the authors.)

107

ENZYME CHARACTERIZATION

the fully automatic method is to use a suitablepH gradient, monitored by a continuous-flow p H meter. One of the difficulties is that the buffering capacity of the various components of the reaction mixture may vary with pH. It may therefore be difficult to predict the p H precisely during the gradient. Probably the best approach is to calibrate the pH-gradient-making system with all reagents present before performing the actual enzyme assay. An example (fig. 7.3) of the automatic determination of the pH optimum of an enzyme is given on 8-glucuronidase (Tappel and Beck 1965). Barrera et al. (1969) have determined the pH optimum of ribonuclease with a p H gradient in the autoanalyzer.

7.3. Enzyme stability The stability of enzymes either on storage or at various temperatures is readily studied, simply by continuous-enzyme monitoring. Fig. 7.4 shows the relative stability of yeast L( +)-lactate dehydrogenase at 0 110

I

I

-

30

-

20

-

10

-

0

0

-

I

I

25 50 Incubation time of enzyme (min)

75

Fig. 7.4. Automatic determination of enzyme stability. Yeast mitochondria1 L(+)lactate dehydrogenase assayed continuously at 37 "C. Enzyme kept at 0 or 40 "C during the assay. (From Roodyn 1969.) Subject index P.

218

108

AUTOMATED ENZYME ASSAYS

and 40 "C,measured by keeping the enzyme in a temperaturecontrolled vessel with gentle stirring and pumping at a slow rate continuously into the analyzer.

7.4. Temperature optimum Temperature optimum may be determined automatically by continuously altering the temperature of the reaction vessel during the assay, preferably using a temperature recorder. In the Technicon heating baths, if the heaters are switched on with the baths at room temperature, the increase in temperature is sufficiently slow and steady for measurements of temperature optimum to be made over a convenient period of time. Tappel and Beck (1965) reported that the heating bath warms up almost linearly at about 1 "C/min, but I have found that the deviation from linearity is significant. An example of automatic measurement of temperature optimum is given in fig. 7.5 with yeast L(+)-lactate dehydrogenase. If the data in this figure over the range 19 to 35 "C are plotted as an Arrhenius plot, good linearity results (fig. 7.6). Thus, the analyzer is of use in measuring the energy of activation of enzyme reactions. (Tappel and Beck (1965) have published a similar plot for 8-glucuronidase, and Barrera et al. (1969) for ribonuclease.)

7.5. Plots of multi-parameter studies I have mentioned the use of 'enzyme gradients' in M.E.A. in order to overcome the difficulty of assaying groups of enzymes at one protein concentration. The enzyme gradient is another example of an automated method of study of enzyme properties (fig. 7.7). The automatic measurement of the progress curve has been discussed fully (0 5.3). Thus, all of the important measurements in initial kinetic studies on enzymes may be automated. This situation should stimulate investigation into new methods for plotting or tabulating multi-parameter results. For example, it is relatively easy with a dual-channel analyzer and enzyme gradient to obtain simultaneous plots of enzyme activity

ENZYME CHARACTERIZATION

109

Fig. 7.5. Automatic determination of effect of temperature on enzyme activity. Assay for yeast L(+)-lactate dehydrogenase. The heating bath was set initially at 20 OC and allowed to heat up slowly while the enzyme and reaction mixture were pumped through the analyzer. (From Roodyn 1969.) 2.335 OC

.-.wx>

'G 2.2 U a

E

3 C Ic

0

g

-I

2.1 -

19 "C

I I 2.o 3.2 3.3 3.4 Reciprocal of absolute temperature xIO' Fig. 7.6. Arrhenius plot of data in fig. 7.5. The logarithm of enzyme activity was found to vary inversely with the reciprocal of the absolute temperature over the range 19 to 35 "C.

Sublecl index P. 218

110

AUTOMATED ENZYhE ASSAYS

Fig. 7.7. Automatic determination of relationship between enzyme activity and protein concentration.Assay with L(+)-lactate dehydrogenase of yeast. The protein concentration was increased linearly, and was directly proportional to cup number. (From Roodyn and Maroudas 1968.)

against time and protein concentration. Such results may conveniently be plotted as three-dimensional diagrams, in which surfaces of linearity may be defined. Two plots of this nature are given in figs. 7.8 and 7.9; The first is satisfactory, but the second shows serious deviation from linearity. A hypothetical plot (fig. 7.10) of substrate concentration, enzyme activity and incubation time is an indication of the sort of diagram one might obtain with further kinetic studies. Recently, by using a variable-speed pump, it has been possible to determine the relation between enzyme activity and protein concentration for a

ENZYME CHARACTERIZATION

Time of incubation (min)

Fig. 7.8. Three-dimensional plot relating incubation time, protein concentration, and enzyme activity. Assay with D(-)-lactate dehydrogenase of ‘petite’ yeast. (From Roodyn 1967a.)

Time of incubation (min)

Fig. 7.9. Three-dimensionalplot as in fig. 7.8. with severe deviation from linearity. NADH-ferricyanidereductase of yeast. (From Roodyn 1967a.) Subiccr index P. 218

AUTOMATED ENZYME ASSAYS

112

K Incubat ion time

Fig. 7.10. Hypothetical three-dimensional plot relating incubation time, substrate concentration and enzyme activity. Graphs for total activity drawn with three incubation times and six substrate concentrations. The value for Km at each time is indicated by ‘X ’ and the average value would be obtained by drawing the best line through these values to give an intercept on the substrate concentration axis (‘L’).

A

0

2

L

6

10

0

Incubation time (min)

Fig. 7.1 1. Three-dimensional plot with 6 incubation times. D(-)-lactate dehydrogenase of yeast assayed in a variable-speed enzyme analyzer. The assay is linear with the respect to protein concentration and incubation time over the surface ABCD. (From Roodyn 1969.)

series of incubation times, giving a more detailed three-dimensional plot (fig. 7.11.). Figs. 7.8, 7.9 and 7.11 were in fact obtained from multi-enzyme analyzers, and a family of such curves was obtained in each assay. Space does not allow inclusion of the full output of a typical assay using such systems, but it is clear that the automated technique is capable of giving a wealth of data. Fully automated systems will

ENZYME CHARACTERIZATION

113

doubtless be developed which will routinely perform the standard characterization measurements discussed in this chapter on large numbers of enzyme samples. Whereas it is usual to obtain a single value for enzyme activity, it may not be unusual in the future to obtain a detailed ‘profile’ on the enzyme, giving its K,, pH optimum, temperature optimum, stability and other properties, each value obtained from many measurements of the progress curve and of the relation between activity and protein concentration.

Subiecr index P. 218

CHAPTER 8

Calculation of enzyme activities from instrument readings

In this chapter I will discuss the last stage in enzyme automation, namely the processes involved in the conversion of instrument readings into suitable units of enzyme activity. These calculations involve actual processing of the data e.g. transfer from a record on the chart of a pen recorder to a computer, measurement of certain properties of the analyzer, such as retention time and flow rates in the various components, and finally conversion of the readings in a given set of assays to suitable units, using data obtained from the instrument calibration. It is convenient to consider the data processing separately, and the instrument calibration and calculation of results together.

8.1. Data processing There is little advantage in developing automated assays if the data cannot be processed at the same rate as the output from the analyzer. The actual form in which data are required varies from one laboratory to another. In some cases, the actual record on the chart of the pen recorder is adequate (for example in semi-automated methods) whilst in others a standard ‘form’ is required of the results in suitable units (for example the patient’s record in a clinical laboratory). The commonest machine output is still the chart of a pen recorder, although there is frequently a digital paper tape output in addition. The pen recorder is essentially an analogue device, providing a continuous record of change in the reaction mixture as followed by the sensor. In 114

CALCULATION OF ENZYME ACTIVITIES

115

order to convert an analogue trace into meaningful units the ordinates and abscissae have to be calibrated. For example, the chart speed may be determined to convert movement of the paper into units of time, and the response of the sensor to different concentrations of standard solutions is measured in order to convert movement of the pen into units of concentration. If the parameters measured behave linearly in both the X and Y axis, all that is required is to calculate appropriate conversion factors for each axis and then use the analogue record directly. For example, if the instrument response is proportional to absorbance, if the substance under study obeys the Lambert and Beer law and if the chart movement is directly proportional to time, a trace of a progress curve on a recording spectrophotometer may be directly converted to absolute units simply by altering the scale divisions. Unfortunately the situation is rarely as simple as this. The instrument response may not be proportional to concentration over its entire range and the variable along the X axis may not be time but sample number or p H . With irregular sampling or a non-linear pH gradient it may not be possible to calculate a simple conversion factor for this axis. Also, sometimes the output may be digital in essence, although apparently analogue in form. In the common case of an autoanalyzer measuring some component in a series of samples, the value of interest is not the shape of the sample peak, but the steady state value at each peak height. The instrument is in fact printing a series of digital values that have to be measured and suitably processed. This processing may be performed by visual inspection, and several workers have described very simple graphical methods for reading autoanalyzer charts. For example, Allen (1966) used a T square attached to a vertical rule sliding over the recorder. The rule was moved up to the peak and the value converted into absolute units from a series of calibration curves drawn along a strip of recorder paper attached to the top of the instrument. I have found that it is not too tedious to transfer the peak values manually either onto suitable coding forms for further computer processing, or directly into an IBM card punch. However, the burden of doing this could well be excessive in multi-channel systems. Recently Maltby (1968) has described a system for reading Subjcrl index P. 218

116

AUTOMATED ENZYME ASSAYS

autoanalyzer charts that may be of general use. The chart is attached to a trace-reader (Normalair-Garrett Ltd., Yeovil, Somerset) which is essentially a steel sheet with a 1 V potential difference across the vertical axis. The operator presses a probe against the chart at a selected point (e.g. the top of a peak), and the voltage, proportional to the distance of the probe along the Y axis, is passed to an analogue-todigital converter and thence to an Olivetti Programma 101 desk-top computer. The method has the great advantage that the operator can select peak heights, reject spurious peaks and generally edit the output from the sensor. Computer programs to perform these functions would necessarily be rather large. It is also an advantage to have some human intervention in the chain of events between introducing the reaction mixture into the sensor and obtainingthe finalcalculatedvalue. (A chart reader system giving digital output is also marketed by P.C.D. Ltd., Farnborough, Hampshire.) If one wishes to automate the data processing completely, however, one has to have some system for directly pi ocessing the output of the sensor. A variety of methods of different degrees of complexity have been described to achieve this. Many commercial spectrophotometers have systems for direct digital read-out, usually on paper tape (see appendix 111 for further details). In the interrupted flow system of Pitot et al. (1968) described in 4 4. l., the enzyme analyzer was connected to a digital voltmeter with system control, and hence into an output adapter feeding into an IBM card punch machine. The punched cards were then processed off-line by computer. Various electronic systems for on-line computation of results have also been described. For example Kindig et al. (1967) describe an ‘electronic slope reader’ which calculates continuously the rate of change of absorbance with time, using the output from a recording spectrophotometer. In the method of Neese and Mather (1967) a Gilford 2000 multiple-sample absorbance recorder is connected to a computer. The operator follows the progress of the reaction and selects a linear region of the progress curve. The computer takes two points from this region, corrects for sample dilution and gives an immediate print-out of enzyme rate. Bernier et al. (1966) describe the use of a small electronic calculator

117

CALCULATION OF ENZYME ACTIVITIES

to interpolate readings into a standard curve and hence calculate the concentration of an unknown sample. Regression analysis is used in the calculation. Programs for data processing of enzyme analysis on the Olivetti Programma 101 are given by Borner (1968). Technicon have produced various data logging systems for such operations as peak recognition, base-line correction and calibration by standards. The operation of the Technilogger I1 is as follows. A signal conditioner takes the analogue signals from the recorders and transforms them into voltages that are scaled and proportional to concentration of the component under assay. The scanner or multiplexer passes the output signals of the signal conditioner one at a time to a digital voltmeter. The data, now in digital form, are suitably formatted and passed to an output device such as a line printer or paper tape punch. A master control unit supervises the operation of the whole system. Several detailed descriptions of computer-based automated assay systems have now been published. Many deal with general problems of data processing and record keeping, particularly in the clinical laboratory. A useful example of the arrangement of such a system is given by Gould (1967). There are many ways of automating the processing of data, and Farr et al. (1966) discuss the relative merits of online analogue and digital computing, and off-line batch digital computing. They describe a relatively simple analogue system which includes a digital voltmeter and which interpolates into a calibration curve so as to give a digital print-out of results in units of concentration. Wootton ( 1968) also discusses computer-based systems. The usual method is to connect either the recorder or the sensor itself to a suitable analogue-to-digital converter, and thence either directly to the computer (as with the system of Evenson et al. (1967) using the LINC computer) or to a paper tape, card punch or magnetic tape recorder. The physical record so produced can then be processed offline. Various programs have been written to evaluate data in this form. Gould (1967) describes an on-line system which corrects for drift in the base-line as well as for changes in the sensitivity of the assay. The peaks are recognised and determined by three processes: slope of the signal, the time from the last peak, and the duration of the peak. Subircr index

n. 218

118

AUTOMATED ENZYME, ASSAYS

Gould points out that measurements based on one parameter only, such as time between peaks, are not necessarily reliable. It is important to have a record of the shape of the peak, in addition to the final digital value of concentration. For example, if the operator only half fills a sample cup, a simple digital output will give no indication that an error has occurred, whereas the analogue record would show an unusually sharp peak. A similar problem arizes in the SMA/12 (Sequential Multiple Analysis system) in which a multi-channel analyzer is ‘phased’ to give a record on a single output device (see Q 6.4.) At any particular time only a small portion of the peak in each channel is being recorded; and errors in the channel may be missed. Smythe et al. (1967) discuss this problem and describe the use of a multistage storage oscilloscope which gives a continuous record of the changes in concentration in each channel. I hope that the above brief account will give the reader an indication of the various approaches to the problem that have been adopted in different laboratories. The subject is in a state of very rapid development and it is impractical to give a comprehensive treatment of it here. Payne (1968) gives a concise account of the history and basic principles of computer technology. I have included a detailed description of the use of a typical Fortran program in Q 9.3 and appendix IV. This, I hope, will illustrate the extraordinary flexibility and usefulness of such methods when married to automated assay techniques.

8.2. Calibration and calculation of results The following treatment was developed for use with the Technicon autoanalyzer and is particularly suited for continuous-flow systems. The theory of operation of such analyzers is discussed in detail by Jonnard (1960) and the precision and inherent errors of the method assessed by Thiers and Oglesby (1964). I will endeavour, however, to treat the subject as generally as possible and most of the formulae should be relevant to other systems, such as discrete-sample analyzers. In Q 9.3 and appendix IV, I describe a computer program for calculation of results from the calibration data and experimental readings and

CALCULATION OF ENZYME ACTIVITlES

119

blanks. The treatment below follows the main sections of the computer program, and uses variable names that are similar, where possible, to those in the program. The reader conversant with Fortran should therefore be able to follow the detailed working of the program. The definition of the variable names used in the computer program is given in appendix IV.3, whilst the terms used in this chapter are defined in the text when first mentioned. 8.2. I . Molar conuersion-factor calculation Perhaps the commonest method of following enzyme reactions is to measure changes in the absorption of light. The usual method of converting absorbance readings into molarities is to refer to a previously measured molar extinction coefficient. Assuming that the Lambert and Beer relationship holds, if a chromophore has a molar extinction coefficient of E and the cell has an optical path of 1 cm, the concentration of that chromophore (CC) in pmoles/ml that corresponds to an instrument reading of R and a blank of B is given by:

C C = ( R - B ) ~ 103/&.

(8.1)

If the optical path is not 1 cm, but is some other value OP, the above relation is modified to :

cc = {(R-B)

x 103/4 x OP.

(8.2)

Sometimes the value of OP is stated by the manufacturers of the cell. However, in many continuous-flow colorimeters it is more important to have minimum retention in the flow cell than optically flat surfaces placed a known distance from each other. In the Technicon tubularflow cells, the path length of light is increased by internal reflectance and is in fact greater than the physical length of the cell. The optical path may therefore not be identical to the length of the cell, and is best determined experimentally by placing a solution of known absorbance in the flow cell. In general it is unwise to rely on published molar extinction coefficients unless one is absolutely certain that the instrument used in the automated assay gives the same readings with standard solutions as the instrument used to determine the published value. Subject index P. 218

120

AUTOMATED ENZYME ASSAYS

Not all chromophores obey the Lambert and Beer law, and not all colorimeters give readings that are linearly proportional to absorbance. Also, many assay systems are based on parameters other than changes in light absorbance. It is therefore useful to have a more general term than molar extinction coefficient for the calculation of results. I have found that a convenient value (see ch. 5 ) is the ‘molar conversion factor’ which is defined as that concentration of solute in pmoleslml that gives an instrument reading of 1.0. This general definition may be applied to any analytical system capable of measuring the absolute concentration of a substance. If the instrument is a spectrophotometer, the solute is a chromophore obeying the Lambert and Beer law, the light path is 1 cm, and the instrument readings are directly proportional to optical density, MCF (molar conversion factor) = lo3/&

(8.3)

In general, the concentration of solute (CS) in pmoles/ml corresponding to an instrument reading of R and a blank of B is given by:

CS

= (R-B)*MCF.

(8 -4)

If the response of the instrument is linearly proportional to solute concentration this will apply to all readings. In the computer program below, the value of MCF is calculated for a series of concentrations. As soon as any value differs by more than 5% from the previous value, all previous values are averaged to give the average molar conversion factor (AMCF) for the linear region of the calibration curve. Any reading outside this range is rejected, with a suitable warning notice. This system works well for most colorimetric assays, where the instrument response is usually linear over the range 0-1.0 absorbance units. The value for AMCF calculated above takes into account any losses of material that may occur during the analysis. For example if a dialyzer is used, perhaps only 10% of the solute may pass across the membrane. The conversion factor therefore refers to the result obtained after putting a known amount of solute into the analyzer and not to the characteristics of the chemical reaction itself. For example, a

CALCULATION OF ENZYME ACTIVITIES

121

reading of 1.O may be equivalent to 0.8 pmoles/ml of potassium ferricyanide when pumped directly through the flow cell, but to 8.0 pmoles/ ml if the dialyzer is used. Thus, wherever possible, the analyzer should be calibrated by internal standards subjected to the same sequence of treatments as the unknown sample. There are some situations in which the calibration curve is far from linear and a single conversion factor cannot be used. A common cause of this is when a photometer that reads in per cent transmission (% T) is used, rather than one that measures absorbancy (2- log,, (yoT)). The easiest approach in this case is to convert the yo T readings into absorbancies. A useful and clear table for doing this is given by Briggs and Connerty (1967). McKay et al. (1964) describe an electronic system for converting the yoT value of a colorimeter into a linear value, and many recorders can be ‘linearized’. If a linear relationship cannot be obtained, it is necessary to determine the concentration of unknowns by interpolation into a standard calibration curve. This may be done graphically by drawing the standard curve on a transparent surface and sliding it over the trace until the unknown crosses the curve. These ‘trace readers’ are commonly used in Technicon systems. Linear interpolation may also be done by computation, preferably in a computer. The basis of the linear interpolation method is as follows: If a reading (R) falls between the ith and j t h readings on the calibration curve (CR, and CR,), corresponding to concentrations of solute of CSi and CS, respectively, the concentration of solute at this reading (CS,) is given by: CS, =CSi+(CSj-CSi)(R-CRJ/(CRj-CRJ.

(8.5)

(This assumes that the reading increases with solute concentration.) More complex treatments take into account the instrument zero, which should be subtracted from all readings and blanks, and the blanks with standard an unknown. The most precise treatment is to correct each reading of the calibration curve for its own blank. If the corresponding blanks in the above treatment are B, CB, and CB,, the value (R - B) should fall between the values (CR, - CB,) and (CR, - CB,) and eq. 8.5 should be appropriately corrected. If the blanks are Subject index P. 218

122

AUTOMATED ENZYME ASSAYS

reasonably constant throughout the assay, it may be sufficiently accurate to determine initial and final blanks only, and to calculate all other blanks by linear interpolation. (This facility is provided in the program below .) It is of course possible to calculate molar conversion factors not only from readings on pen recorders, but also directly from the output of the sensor. For example, in the particular case of colorimetric analysis, Abdullah (1967) discusses the relation between chromophore concentration and voltage. He presents a computer program to calculate concentration from punch tape output of a digital voltmeter connected to a colorimeter. In a dual beam colorimeter (for example in the Technicon system), if the sample photocell is read against the reference photocell and the output of the photosensitive device is linear, then 1 0 g ( V ( ~ b ) / ~ (=k.CS.OP, ~s))

(8.6)

where V(Z,,) and V(ZJ are the voltages with incident light, I b and Z,, from the blank and sample respectively, k is a constant related to the molar extinction coefficient, CS is the concentration of solute, and OP is the optical path. With the development of on-line computers connected directly to the sensor, this approach will become more common. 8.2.2. Incubation time For interrupted-flow methods, the incubation time (1T) is directly proportional to the distance along the trace and may be calculated directly from the chart speed, In a discrete-sample analyzer the time is the interval between addition of enzyme to the reaction mixture and entry of the latter into the sensor. It is determined by the rate of movement of the sample train and the distance between the enzyme probe and the sensor. Similarly, in continuous-flow systems, the time of incubation is the interval between addition of enzyme to the reaction mixture and entry of the latter into the flow cell of the sensor. In this case the time is determined by the volume of the incubation vessel and the total rate of flow of fluid plus gas. If the volume of the

CALCULATION OF ENZYME ACTIVITIES

123

reaction vessel between the entry point of the enzyme and the sensor is V ml and the total flow rate of fluid plus gas is F ml/min, the incubation time is equal to V/F. V may be determined by filling the vessel to the appropriate point and pumping the contents into a measuring cylinder, but it is difficult to obtain a precise measure of F if a gassegmented stream is used. If the volume of gas pumped per minute is G ml, and the total line volume of the fluid lines is TLV ml/min, we have IT = V/(G+TLV).

(8.7)

Unfortunately, the value of G will depend on the temperature of the bath and the pressure in the reaction vessel. I have found that the effect of temperature on IT is somewhat greater than would be expected from simple expansion of the gas (see fig. 5.2). It is possible that the viscosity of the fluid falls as the temperature rises so that the pressure in the vessel changes. It is thus difficult to obtain a precise measure of incubation time from the expected flow rates in the gas and fluid lines. A more convenient method that may be used in both continuousand discrete-sample analyzers is to introduce a suitable marker while the instrument is in operation (see Schwartz et al. 1961). Marks are made on the chart after a known interval in order to measure the chart speed, and also when the added marker enters and leaves the incubation vessel. The last event usually corresponds to the entry of the marker into the flow cell. If the distance between marks for incubation time determination is DMIT, the distance for chart speed determination DMCS and the time between marks for chart speed determination TMCS, IT =DMIT*TMCS/DMCS.

(L apologise for the rather complex variable names. As mentioned above, where possible the names here correspond to those in the program in appendix IV). For a precise estimate of the effective incubation time, the time involved in mixing enzyme and reaction mixture should be taken into account (see § 5.2). However, if the total Subject index P. 218

124

AUTOMATED ENZYME ASSAYS

time of incubation is reasonably long (Lea, 5 min or more) correction for mixing is generally small and may be ignored (See Schwartz et a]. 1961, and fig. 5.1). 8.2.3. Line-volume calculations A ‘line’ is any device for introducing or removing fluid from the analyzer. In the Technicon system it corresponds to the pump tubing and attached reagent lines. In discrete-sampling systems it corresponds to the reagent probe or syringe. The determination of line volumes (or more precisely line flow rates) is best done with marker solutions fed into the analyzer while it is operating under its usual conditions. For example, it may be inaccurate to measure line volumes in the Technicon proportioning pump by disconnecting each line from the manifold and measuring its flow rate by collecting fluid in a measuring cylinder, because disconnection of lines will alter the pressure in the flow system and hence the relative flow in different lines. Also the juxtaposition of pump tubing of different diameters can have serious effects (fig. 8.1), Position 2 Large tube

Position 1

Platen

Fig. 8.1. Possible error arising from action of Technicon proportioning pump. The pump operates by a series of moving bars compressing a row of plastic tubes. If a wide and narrow tube are next to each other a pumping error may result as follows: In position 1 both tubes are compressed. In position 2, however, the small tube remains open and fluid is therefore not pumped through it. As a result the relative rates of pumping in the two tubes varies with the time in the pumping cycle, giving erratic results. (From Smythe et al. (1967), by courtesy of the authors.)

CALCULATION OF ENZYME ACTIVITIES

125

so that the flow must be tested with the lines in their normal position. Fractional line volume (FLV) is the flow rate in a line divided by the total flow rate of all fluid lines making up the reaction mixture. This excludes gas lines, waste lines and any gradient lines that do not flow directly into the reaction mixture. The total line volume (TLV) is measured by collecting the fluid output from the incubation vessel in a measuring cylinder for a fixed time. The instrument is calibrated by running markers into each line separately. Provided that all readings and blanks are in the linear region, the fractional line volume of the ith line is given by FLVi=(LRi-LBi)

(8.9) 1

where LB, LR and n are the line blanks, line readings, and number of lines, respectively. If the line readings or blanks do not fall in a region in which the instrument response is proportional to marker concentration, appropriate conversion factors must be made as in tj 8.2.1. The observed line volume (LVO) is then calculated from the product of the fractional line volume and the total line volume, and compared to the theoretical line volume (LVT). The line accuracy may then be estimated and the value used to determine the precise composition of the reaction mixture. 8.2.4. Calculation of concentrations of line reagents Once the lines have been calibrated, it is possible to calculate the concentration of each reagent to be pumped by a given line to give the desired final concentration in the reaction mixture. Let us assume that the concentration of the component in a suitable stock solution is CCS, the volume of line reagent to pump that component required for a group of assays is CVL and the concentration of component required in the reaction mixture is CCRM, then the volume of stock solution of the component (CVSR) to be made up to the volume CVL is given by

CVSR =CVL*LDF*CCRM/CCS,

(8.10) Subject index I. 218

126

AUTOMATED ENZYME ASSAYS

where LDF is the line dilution factor of the line carrying that component. If the sampling rate is S/hr, and the number of samples in the assay is NS, C V L= N S . LV O . . ~ /Sml.

(8.1 1)

These equations can be used to calculate precisely the volume and concentration of various reagents required. In small experiments this is not very important. In multi-channel systems operating for prolonged periods of time, however, it is important to make accurate estimates of these parameters in order to maintain a steady supply of reagent and also to make accurate estimates of cost. If the stock solutions are prepared at exactly ten times the final concentration required in the reaction mixture, the volume of stock required per 10 ml final volume of line reagent is equal to thelinedilution factor. This may be calculated from marker readings as described above. However, a rapid way to check the dilution factor is to connect the line to a burette and measure the time required to empty a specified volume(G. K. Jowett, personalcommunication). For example, if the final substrate concentration required is 2.0 mM, the substrate line pumps at a rate of 0.62 ml/min and the total line volume is 3.1 ml/min, the LDF is 3.1/0.62 or 5.0 and the line reagent may be prepared by diluting 5.0 ml of 20.0 mM substrate to 10 ml. At present such dilutions are done manually. However, in the future the preparation of line reagents from stock solutions may be performed automatically under the control of an on-line computer, which uses data on line calibrations previously fed into it. 8.2.5. Gradient calibration and gradient-making systems The subject of gradient-making systems in analytical chemistry is a very large one and space does not allow a full treatment. However, it should be stressed that the combined use of autoanalyzers and gradientmakers has great possibilities and is as yet in a very early stage of development. One may determine the shape of a gradient by theoretical considerations, iterative computations, preferably on a computer, or by direct measurement with appropriate marker solutions. I will only

127

CALCULATION OF ENZYME ACTIVITIES

consider the relatively simple gradient-making system i n which fluid is pumped into and out of a single mixing vessel. Calculation of gradients with two mixing vessels is given by McGilvery (1960) and the theory of multi-chambered gradient systems has been discussed in detail by Peterson and Sober (1959), by Peterson in this series of monographs, and by Sorin and Vargues (1966). Burns et al. (1965) used an analogue computer to simulate a nine-chambered gradient maker and hence to compute gradients, including pH gradients. Computer analysis of multi-chambered gradient systems is also given by Pitt and Scott (1969). The reader is referred to these references for a fuller treatment of the subject. For a single vessel, if the rate of filling is x ml/min, the rate of emptying y ml/min, the volume of liquid in the vessel is initially V ml, the concentration at zero time is C,, and at time t min is C , and the concentration of fluid pumped into the vessel is C,, Sorin and Vargues (1966) have established the following relationship :

C,=C,+(C,-C,)

[

1

x-y)t + (-

y--x

v l

(8.12) *

In the case where C , =O and y =2x, CJC, =GDF, =yt/2V,

(8.13)

where G D F is the gradient dilution factor. Thus if we pump a solute into a mixing vessel initially containing water and pump out a t twice the rate of filling, we obtain a linear gradient, whose slope is proportional to the rate of emptying and inversely proportional to the volume of fluid initially in the mixing vessel. This is confirmed experimentally in Figs. 8.2 and 8.3 (see also Davis et al. 1965). If x = y in the above system the volum: of fluid in the mixing vessel remains constant throughout the gradient and a logarithmic gradient is obtained. Schwartz and Bodansky (1966) dripped water into a stock solution in a mixing vessel and pumped out at the same rate. They gave the rate of dilution as ((V-x)/x}'C,. For similar systems see Subiecr index P . 218

128

AUTOMATED EYZYME ASSAYS

-

a,

ln

a,

>

.-C

I

5

Time (min)

Fig. 8.2. Effect of volume of fluid in mixing vessel on gradient. Vessel filled at 0.32 ml/min and emptied at 0.6 ml/min. Volume of fluid initially in mixing vessel varied as shown.

Volume in m i x e r

(rnl-')

Fig. 8.3. Plot of rate of formation of gradient against reciprocal of volume in mixing vessel. (Data from fig. 8.2.)

Posen et al. (1967), and Duggan and Gurll (1965). One can obtain gradients other than linear or logarithmic by varying the ratio of x to y . Some examples of such gradients determined empirically are given in fig. 8.4. Returning to the linear gradient in eq. (8.13), it is more convenient to relate gradient dilution factors to sample number than time. For a

CALCULATION OF ENZYME ACTIVITIES

129

I I I

I /

/

,E ,,

Rate of emptying

Rate of filling 0.6

I

/

2.o

7.5

00

10

20 30 Cup number

40

Fig. 8.4. Effect on shape of gradient of ratio of rate of filling of mixing vessel to rate ofemptying. The gradients in this figure (and in figs. 8.2 and 8.3) were followed by a ferricyanide marker solution, measured at 420 nm. The time of operation of the gradient was related to cup number of the sampler. The ratios refer to the relative rates of filling and emptying of the gradient making vessel. (From Roodyn and Maroudas 1968.)

component in the stock solution of concentration CCS being pumped into the gradient mixer and thence into the analyzer by a gradient line of fractional line volume FLV,, the concentration of component in the reaction mixture (CCRM) is given by CCRM =CCS * FLV; GDF.

(8.14)

Combining eqs. (8.13) and (8.14) the concentration in the reaction mixture for sample number SN with a sampling rate of S/hr is given by CCRM SN =CCS * FLV, * LVO; SN. 30/S. V,

(8.15)

Sublecl index p . 218

130

AUTOMATED ENZYME ASSAYS

where the rate of flow from the gradient maker to the analyzer is LVO,. Since FLV, is equal to LVOJTLV, where TLV is the total line volume, we also have CCRMsN=CCS * LVO;

- SN

*

3O/S * V *TLV.

(8.16)

With a linear gradient system of the sort described, therefore, the concentration of a component in the reaction mixture for any given sample is proportional to the square of the flow rate from gradient maker to analyzer and inversely proportional to the volume of fluid in the gradient maker and the total fluid flow through the analyzer. Taking an actual example, the gradient line volume was 1.08 ml/min, the total flow rate 4.06 ml/min, the sampling rate 40/hr, the initial protein concentration of enzyme pumped into the gradient maker was 5.56 mg/ml and the volume of fluid initially in the mixer was 25 ml. The protein concentration in the reaction mixture for sample number 10 should have been 5.56 x 1.08’ x 10 x 30/40 x 25 x 4.06 mg/ml or 0.478 mg/ml and the observed value was 0.482 mg/ml, in good agreement. It is thus possible to use eq. (8.16) to calculate concentrations under different assay conditions. In practice certain difficulties arise. Ideally the gradient should be started at precisely the moment that the first sample flows in the output from the gradient maker. However, because the delay time from sampler to reaction vessel may be different to that from gradient maker to reaction vessel it could be difficult to do this. There is also a certain amount of ‘dead space’ between the mixing vessels and the reagents and some time elapses between connecting the reagents and starting the gradient. Because of errors in the pumping rate, the rate of emptying may not be exactly twice the rate of filling, so that the gradient will not be perfectly linear. For these reasons it is advisable to calibrate the gradient empirically by using a marker. A suitable marker or ‘stock’ solution is first run directly into the analyzer giving a reading (‘stock reading’ or SR) The marker is removed and the base line measured (‘stock blank’ or SB). The mixing vessel is then filled with the appropriate volume of water and the gradient is run by pumping stock solution into it. A trace is thus ob-

CALCULATION OF ENZYME ACTIVITIES

131

tained of ‘gradient reading’ or GR against time, and after the appropriate base-line (or ‘gradient blank’, GB) has been measured for each value of GR, the gradient dilution factor for any point on the trace may be calculated from the expression :

GDF =(GR- GB)/(SR- SB).

(8.17)

The value corresponding to a particular sample may be calculated from the sampling rate, the sample number and the time elapsed since the start of the gradient. However, it is more convenient to identify the position corresponding to each sample in the gradient by performing the gradient calibration with the sampler operating and with water in each cup. As each sample is aspirated a small trough appears in the trace (see fig. 9.9). 8.2.6. Protein concentration The reader is referred to Thurman and Boulter (1966) for a critical account of current methods for measuring protein concentration. This concentration in the reaction mixture must be calculated if the specific activity of the enzyme is required. It is equal to the product of the protein concentration in the enzyme line (PCEL) and the fractional line volume of that line (FLV,). If the enzyme is not pumped directly into the analyzer but is pumped first through the gradient-making system, then the protein concentration in the reaction mixture (PCRM) for sample number SN is given by:

PCRM =PCEL.FLV, * GDF,N.

(8.18)

If different samples of enzyme are used, there would be a different value of PCEL for each sample. 8.2.7. Sample pattern and theory of sampling The sample pattern is the arrangement of samples in the sampler unit. For enzyme sampling it would consist simply of the unknowns. In multiple-enzyme analysis however, it is a specifically designed arrangement of groups of substrates. The concentration of a sample in the reaction mixture is equal to Subject index P. 218

132

AUTOMATED ENZYME ASSAYS

the product of its concentration in the sample cup and the fractional line volume of the sample line. However, this is only so when the sample concentration has reached steady state. This is best understood by consideration of the nature of the sampling process. This may be illustrated as follows. A flow system is set up with all lines in water. The sampler probe is then transferred rapidly from water to a suitable marker. After a time it is returned rapidly to water. A typical trace is shown in fig. 8.5. (see also Smythe et al. 1967). The curve consists of three parts: a rise curve, the steady state curve and a fall curve (see Thiers et al. (1966) for recommended terminology). The perfect analytical system would give a square-topped curve, as shown by the dotted line. In practice, the sample concentration approaches steady state in a finite and measurable time, and after transfer of the sample probe to water it falls to its new steady state (zero concentration) in a similar way. The shape of the rise and fall curves may be measured by placing Steady sta’te 0 A

P

5 :”

0 0

R

I!

Fig. 8.5. Typical trace observed on transferring probe from water to marker and back. Ferricyanide marker used, with system described in ch. 9. The probe was attached to the sample line.

I

0

I

30 60 Time of sampling (sec)

I

90

Fig. 8.6. Relation between sampling time and approach to steady state. Results with the system described in ch. 9, using a total fluid flow rate of 6.1 ml/min.

Fig. 8.7. Expected shape of sample peak from sampling system given in fig. 8.6. The curves were calculated assuming that the fall curve follows the same dilution laws as the rise curve.

134

AUTOMATED ENZYME ASSAYS

the sampler probe in the sample for increasing times (e.g. 5, 10, 15,20 sec etc.) with a wash between each time. The trace obtained gives a series of peaks, which are measured to give the relation between sampling time and approach to steady state (fig. 8.6). If we assume that the rise curve follows the same laws as the fall curve, we can calculate the expected shape of the sample peak after different times of sampling, and also the residual amount of sample in the system at various times after sampling was stopped. These calculations were performed with the data in fig. 8.6., and it is seen in fig. 8.7 that a clear plateau in the sample peak would not be visible until 60 sec of sampling. The shape of the sample peak can, of course, be determined experimentally, and an example is given in fig. 8.8. In

-9

0

c.

0

E”

3

>

l

L

Fig. 8.8. Shape of sample peaks at different sampling speeds determined directly. Results obtained by sampling at rates of 70,60, 50,40,30and 20 per hour respectively, from left to right.

CALCULATION OF ENZYME ACTIVITIES

135

practice it is not necessary to reach complete steady state before taking the next sample, and most analytical systems accept some sort of compromise in order to have a reasonable sampling rate. For example, in fig. 8.8, sampling a t 70/hr would give acceptable results even if the peak is not quite at steady state. Errors due to fluctuations in sampling time become greater, however, the further one is from the steady state. Thus in the example in fig. 8.6, a 1 Eec error after 5 sec would change the sample peak from about 40% to 50% of steady state, i.e., a difference of about 20 %. However, a similar error after 40 sec would have no measurable effect on the size of the peak. Sampling error can result from faults in the mechanism of the sampler, but can also be due to variations in the depth of fluid in the sampler cup, particularly if the sampler probe enters the sample rather slowly. In general, therefore, it is better to work reasonably near steady state, although Reid and Wise (1967) have carried out several chemical assays on the autoanalyzer at sampling rates that gave peaks well removed from steady state. They claimed good reproducibility, but mentioned difficulties in wash-out between samples. The shape of the sample peak is directly related to the washing eficiency (WE) of the system. I will define this as the per cent return from steady state to zero concentration in the time interval between samples. The sample carry-over is (100- WE). Thus in our example above (fig. 8.6) the washing efficiency would be 65% for a sampling rate of 6/min, 90% for a rate of 3/min and 99.8% for a rate of l/min. The corresponding sample carry-over values would be 35, 10 and0.2%. Once the wash characteristics of the system are known, one can chose a convenient sampling rate, and also correct samples for crosscontamination. (The analysis of overlapping peaks is discussed generally in ‘Techniques in Amino Acid Analysis’ 1966.) In a train of samples, the reading obtained will be caused by the sample itself and the residual amount of previous sample(s). Thus for sample number SN giving a reading of RSN, the true reading RgN is given by (8.19) The possible error depends not only on the washing efficiency of the subject index P. 218

136

AUTOMATED ENZYME ASSAYS

system but also on the difference in concentration between successive samples. For a given value of WE, one can determine the maximum ratio of readings of two successive samples that would give an acceptable error. For example, with a washing efficiency of 99% and a maximum acceptable error of 5%, the ratio R,,-,/RSN must not be greater than 4.75. Thus if this ratio were only 1.5 we would be well within our desired margin of error, and indeed could operate with a system giving only 97% rather than 99% washing efficiency. To summarize, the error in a reading due to contamination with a previous sample depends on the wash characteristicsof the system, the sampling rate and the ratio of concentration of subsequent samples. Thiers et al. (1966) have made an important study of the sampling process in continuous-flow systems. They made the interesting, and very useful, observation that the changes in concentration from one steady state to another obeyed first-order kinetics. Thus, when the sample probe is moved from sample to water the concentration of sample in solution falls exponentially, i.e., dC/dt = - kC,, where C, is the concentration at time t. If the sample probe is moved from water to a sample that would give a final steady state concentration of C,,, the ‘rise curve’ has the characteristics given by: dC/dt=k(C,,- CJ.

(8.20)

The rise and fall curves were followed experimentally and could be resolved into a lag phase and an exponential phase. The exponential phase could be defined by a term called ‘half-wash time’ or W, which is analogous to the half-life of a radio-isotope and is the time taken to change the concentration at a given time to a concentration half-way between it and the final steady-state concentration. Thiers et al. (1966) give examples of various analytical systems that have been analyzed in this way. For example, an alkaline phosphatase system had a lag of 8 sec and a W, of 21 sec. Their conclusions may be confirmed by fuIther analysis of the results given in fig. 8.6. The lag period was not measured, but if the value log,,(loO-~o steady state) is plotted against time, a straight line is obtained giving a W, of 5.8 sec. The ralue of W, would clearly depend on the flow rate, the faster the flow

CALCULATION OF ENZYME ACTIVITIES

137

Total f l u i d flow r a t e (rnl/rnin) Fig. 8.9. Effect of total fluid-flow rate on half-wash time (W*).System in ch. 9 used at different flow rates. Approach to steady state determined in each case as in fig. 8.6. and W* calculated as described in text.

the better the wash. This is confirmed by an experiment shown in fig. 8.9. Given a knowledge of W, we can calculate what time interval between samples is required to give a required washing efficiency. (One can also express the time in number of samples at various sampling speeds.) For example, if we only require a washing efficiency of 90% we can sample at 20 or 30/hr even with a system that has the very poor half wash time of 60 sec. However, if our analysis demands a washing efficiency of 99.9% (i.e., only0.1% carry over) we need a W , of 40 sec at the most to sample at 20/hr. If we sampled in the same system at 70/hr, we would need a W, of only 5 sec. In the above treatment the component in the sample peak may either be an enzyme (enzyme sampling), a substrate (multiple-enzyme analysis) or a co-factor (enzyme characterization). The shape of the peak will therefore reflect the relationship between enzyme activity (as measured by the response of the sensor) and enzyme, substrate or coSubiecr index p . 218

138

AUTOMATED ENZYME ASSAYS

factor concentration. Examples of effects of these relationships on the shape of the peak have been given in 5 3.3 and it was pointed out that rigorous analysis of the shape of the sample peak may be a rapid way of obtaining useful information about the enzyme-catalyzed reaction. The shape of the peak is also determined by the recording characteristics of the sensor (e.g. maximum rate of pen movement in a pen recorder), and these matters are discussed by Jonnard (1960). Probably most autoanalyzer peaks are not amenable to precise analysis because the recorder used to follow the rise and fall curves had too slow a response. However, it is possible that future experiments with more rapid recording systems will exploit the possibilities of this approach. 8.2.8. Calculation of enzyme activity We now come to the final calculation of enzyme rates. If the reading is R and the blank B, the total reaction (TR) in pmoles/ml substrate transformed (see ch. 1, p. 19) is given by

TR =(R - B) AMCF.

(8.21)

The rate (RA) in pmoles/ml/min is given by RA=(R-B)*AMCF/IT,

(8.22)

where IT is the incubation time and AMCF the average molar conversion factor. The specific activity (SA) in pmoles/mg protein/min is given by SA =(R - B). AMCF/PCRM *IT,

(8.23)

where PCRM is the protein concentration in the reaction mixture. If the protein concentration in the enzyme line is PCEL and the line volume of the enzyme line is FLV,, SA =(R-B)*AMCF/PCEL*FLV;IT,

(8.24)

and if the enzyme line is pumped into a gradient-making system and the gradient dilution factor corresponding to the reading is GDF, SA =(R - B) * AMCF/PCEL. FLY, *IT* GDF.

(8.25)

CALCULATION OF ENZYME ACTIVITIES

139

These calculations assume that the reading R is on the linear portion of the progress curve. If this is not so, a series of values of R and IT must be determined and the true rate calculated either from the slope of a plot of T R against IT or as the intercept at IT =O on plots of RA or SA against IT. Space does not allow a full treatment of the further kinetic calculations that may be made with the results of such rate measurements. Cleland (1963, 1967) reviews statistical methods for analyzing kinetic data and discusses fitting such data not only to the conventional Michaelis-Menten formula, but also to more complex expressions. Thus, if the velocity is v, the maximum velocity V , the substrate concentration S and a, 6, c and d are constants, data may be fitted to any of the three following expressions

and

v = VS/(u+ s),

(8.26)

u = VSz/(d+2bS+ S’),

(8.27)

.

u = V(dS+ S2)/(c+bS+ P)

(8.28)

Cleland (1967) also gives a FORTRAN program for fitting such data by the method of least squares. The statistical treatment of data from enzyme rate measurements is also discussed in detail by Johanson and Lumry (1961). In the above treatment I have considered the various calibrations and calculations in a stepwise manner, and in practice it is useful to perform them in this way. However, it is possible in some cases to combine all relevant expressions given above to produce one rather complex general expression which may be used to calculate a single conversion factor. Multiplication of a given value of (R-B) by this factor would then give the final result immediately. However, this only applies to relatively simple systems with no gradients. Also, in much of the treatment above I have ignored corrections due to instrument zeros and various blanks. A completely comprehensive analysis would take all these into account. However, for most purposes the above treatment should be adequate, particularly if used in conjunction with the computer program in appendix IV. Subject index P. 218

CHAPTER 9

Generalized system for enzyme automation

The previous chapters have shown the variety of approaches that have been used to automate enzyme assays. In this chapter, I describe a generalized system based on the combined use of a Technicon autoanalyzer and the University of London ATLAS computer that is sufficiently simple and flexible to be of use in a large number of enzyme assays. In order to illustrate the principles of instrument calibration and calculation of data described in the previous chapter, I will describe the system in detail, giving a typical experiment as example.

9.1. Flow system The flow system is shown in fig. 9.1. Substrate, buffer, co-factor and enzyme are mixed in that order, and the mixing coil after addition of the enzyme is temperature-jacketed. Since most of the experiments performed with this system have been with M.E.A. the sampler module is connected to the substrate line. However, for analysis of enzyme samples it would be connected to the enzyme line. A sample gradientmaking system is incorporated in the manifold (lines GR and 4, and mixing vessels MI and M,) and is connected to the enzyme line (4). For substrate or co-factor gradients it should be connected to lines 1 and 3 respectively. A variable-speed proportioning pump is used and the speed is controlled by a dial with arbitrary divisions from 0-100. The volume of fluid pumped in the range 30-100 divisions is directly proportional to the number of divisions and the rate of compression 140

Water

Variable speed

P u m p

J MC

Buffer

P S -G

Gas Co-factor

3

Enzyme

Colorimeter

r--7 I I Recorder

7 7 I

I1

(Mixer 21

IMixer 1

I

Fig. 9.1. Flow system of generalized enzyme analyzer. The variable speed pump and gradient-making system allow for selection of different incubation times and protein concentrations (see text). The line volumes are given in table 9.2. D: de-bubbler; G: gas line; GR: gradient line; HB: heating bath; JMC: jacketed mixing coil; pH: pH meter with flow electrode; PS:pulse suppressor; S: sampler; and W: waste.

142

AUTOMATED ENZYME ASSAYS

of the tubes by the rollers (fig. 9.2). The reaction mixture passes into a heating bath, usually operated with a single coil, and thence into a suitable autosensor, usually a colorimeter and pen recorder. It is useful to keep the physical arrangement of the system reasonably undisturbed for different assays and to change the assay conditions by altering the pumping rate, volume of fluid in the gradient mixing vessel (mixer l), composition of the line reagents, sample pattern, incubation temperature, and, with a colorimetric assay, the wavelength of the filters used. For example, although it is possible to change the pump tubing for different assays, it is more convenient to use a standard pumping system as far as possible and to adjust the concentration of reactants in different assays by varying the dilution of stock solutions. The computer program (appendix IV) is used to calculate these dilutions. One can develop ‘families’ of assay systems

-.-

6-

c

E

> E

5-

v

t

-30;

U

-102 .w 3 r

.w

e

030

4b

sb

7b

eb

0

50 90 108g Pump position ( a r b i t r a r y divisions)

Fig. 9.2. Volume of fluid pumped and rate of compressionof tubes at different pump positions. o : tube compressions/min. 0 : volume of fluid pumped. Flow system in fig. 9.1 used.

143

GENERALIZED SYSTEM

based on the invariant physical arrangement of the analyzer. Some of the instrument calibrations will apply to all assays (e.g., the absolute volume of the reaction vessel); others will vary for each assay (e.g., the molar conversion factor for the given reaction). However, even such calibrations may be stored for further assays. For example, calculations of retention times for a particular series of pump speeds may be stored in the computer and called upon where appropriate. In this way it becomes progressively easier to change from one assay to another or to develop new assays.

9.2. Properties of the system Some of the properties of this system will now be given. 9.2. I . Incubution time

With a fixed volume of reaction vessel, the retention time should be inversely proportional to the total flow rate of liquid plus gas (eq. (8.7)). Assuming that liquid and gas are always pumped in the same proportion, the retention time should also be inversely proportional to the total flow rate of liquid. This was found to be true over the range 20 r

-._ c

E

v

?._! c C

0 ._ c m

n

3 U

C

I

a

Pump position (arbitrary divisions)

m

Fig. 9.3. Effect of pumping speed on retention time. Flow system in fig. 9.1 used. Subjrcr index P. 218

144

AUTOMATED ENZYME ASSAYS

tested (fig. 9.3), indicating that the pressure in the system does not build up excessively at high pumping rates. 9.2.2. Fractional line volumes The system was calibrated by marker solutions as described in 0 8.2.3, and the results for the different pump speeds are shown in fig. 9.4. The fractional line volumes were similar at all speeds, with only one line deviating slightly at the lowest speed. This means that the same line 51-

reagents may be used at all speeds to give the desired reaction mixture, so that the time of incubation may be varied in a continuous way simply by altering the speed of the pump. This is a very useful feature of the system, since it enables one to perform a preliminary assay at high speed in order to select a speed that will give readings in the desired range. 9.2.3. Sample interuction and wush characteristics In changing the pumping speed, however, one must not forget that the wash characteristics deteriorate at lower flow rates. This is shown in fig. 9.5 in which the rise curves of the sampling process are shown

145

GENERALIZED SYSTEM

f (4.1)

m

I

0.5 1 Time ( m i d

(5.5)

(4.7)

(6.1)

I

0.5

5-&-

Fig. 9.5. Rise curves at different pumping speeds. Flow system in fig. 9.1 used. The results are expressed as % of the steady-state value. The numbers in brackets refer to the total rate of fluid in ml/min.

for different pump speeds. It is, therefore, important to select a sampling rate that gives the desired resolution a t the pumping speed used. To find a convenient sampling rate for routine work, the carry-over from one sample to the next was measured at a variety of pumping and sampling rates. Between total fluid flow rates of 1.8 ml/min and 5.5 ml/min, it was found that sampling at 40/hr always gave less than 1% cross-contamination. 9.2.4. Gradient-making system In the manifold shown in fig. 9.1., the gradient-making system uses the variable speed pump. As a result, changing the pump speed will change the ratio : volume pumped into mixing vessel volume of fluid in mixing vessel Thus the steepness of the gradient will vary with the pump speed. Since the reaction mixture has the same composition at all pump speeds this provides a suitable way of selecting a range of gradient-dilution factors. Subiecr indexp. 218

146

AUTOMATED ENZYME ASSAYS

The other way to vary the steepness of the gradient is to alter the initial volume of fluid in the mixing vessel. An experimentally determined family of curves of gradient-dilution factor against cup number with 25 ml in the mixing vessel, is given in fig. 9.6. (Similar families of curves have been obtained with a range of volumes in the mixer.)

Fig. 9.6. Plots of gradient dilution factor against cup number at different pumping speeds with 25 ml in mixer. Flow system in fig. 9.1. used, with a sampling rate of 40/hr. The numbers in brackets refer to the pump position. (See fig. 9.2 for the rate of fluid flow corresponding to these positions.)

147

GENERALIZED SYSTEM

We have said above that the incubation time may be varied by changing the pumping speed. This may be done if the enzyme concentration is constant throughout the assay, i.e., if the gradientmaking system is not used for the enzyme. However, if there is an enzyme gradient, changing the pump speed also changes the effective enzyme concentration in the assay, since this is directly related to the gradient-dilution factor. The total reaction observed is proportional to the product of the incubation time and the enzyme concentration. Since these vary in opposite directions, changing the pump speed does not have a dramatic effect on the instrument readings obtained. In fig. 9.7 are shown the effect of pumping rate on steepness of the gradient (curve A) and on the product of gradient dilution factor per cup and the incubation time (curve B). This curve is a measure of the total enzyme activity. If one wishes to vary the incubation time with0.06~

0.04U

\

0

c

U

m .+ c .-

-5 0.02._ -0 Q c .-

U

L Is1

a

0-

I

30

I

I

40 50 60 70 Pump position (arbitrary

80

I

I

90

100

divisions) Fig. 9.7. Effect of pumping speed on gradient dilution factor system in fig. 9.1 used with 50 ml in mixer 1. A gradient dilution factor/cup is the slope of a plot of gradient dilution factor against cup number (cf. fig. 9.6). (A) d gradient dilution factor/cup, at a sampling rate of 40/hr. (B) Product of A gradient dilution factor/cup and incubation time (proportional to the total enzyme activity). Flow rates at various pump positions are given in fig. 9.2. Subircf index P. 218

148

AUTOMATED ENZYME ASSAYS

out changing the enzyme concentration one can either alter the volume of fluid in the gradient-mixing vessel (mixer 1) or, more conveniently, alter the dilution of enzyme in the ‘stock’ vessel (mixer 2). A curve to be used in order to select a suitable dilution of stock enzyme to achieve this is shown in fig. 9.8. Thus one can vary the incubation time, the protein concentration and the type of gradient used very easily with the above system.

‘30

40

50

60

70

80

90

100

Pump position (arbitrary divisions)

Fig.9.8. Method of obtaining constant protein concentration at all pump speeds. V is volume of enzyme to add per unit final volume of fluid in mixer 2 of fig 9.1. The value at pump position 100 is taken as 1.O. Flow rates at the various pump positions are given in fig. 9.2. Values of Vempirically calculated from calibration of gradients (figs. 9.6 and 9.7).

9.2.5. Preparation of reagents The calculations for this are performed by the computer. First of all it is decided which reagents are to be pumped in which lines. The concentrations of suitable stock solutions, the molecular weights of the solutes, where relevant, and the required final composition of the reaction mixture are entered into the computer with the line volume calibrations. Two tables are printed out, one in the form of a weighing sheet for the preparation of stock solutions and the other giving instructions for the preparation of each line reagent from the appropri-

GENERALIZED SYSTEM

149

ate stock solution. Since the pumping characteristics may vary from week to week, the latter table will give different dilution instructions after re-calibration of the instrument.

9.3. Computer program The full program and detailed input instructions are given in appendix IV. Here I will describe the program in a general way, showing how it is used in conjunction with the ‘hardware’ of the autoanalyzer system just described. The program is divided into sections (see appendix IV. 1 for instructions for moving from section to section). They are as follows : 9.3.1. Introduction

General comments of any length about the assay system may be entered here. It is convenient to prepare a detailed account of the system at this stage so that before each group of assays the operator may have precise instructions. At the moment, I have such information stored on cards. However, the most convenient arrangement would probably be to have it stored on magnetic tape in the computer, so that whenever a particular assay or type of assay is requested, full details may be provided. 9.3.2. Molar conversion-factor calculation

For spectrophotometric assays the name of the chromophore, the wavelength used, the optical path and a calibration curve with different concentrations of chromophore are entered here. The program calculates the molar conversion factor for each reading and determines the range of chromophore concentrations over which this factor does not vary by more than 5 % between readings. These values are averaged and used for all subsequent calculations. The maximum value within the linear range is stored and if any subsequent values exceed this a warning notice is printed. The section could be used for any instrument readings in which the concentration of solute is known. Subiecr indexp. 218

150

AUTOMATED ENZYME ASSAYS

9.3.3. Incubation time and chart-speed determinations The instrument is calibrated for chart speed and incubation time as explained in the detailed example given below. These two values are printed out if so required. 9.3.4. Line-volume cahkdtions Details of the total line volume, the expected line volumes and the readings and blanks during calibration are entered here. The fractional line volume, line-dilution factor and observed flow rate are calculated for each line. If the observed value deviates from the expected value by f10% a warning notice is printed. The expected line volumes may either be the maker’s specifications, or more usefully, the values obtained in the previous line calibrations. In the latter case, the warning notice tells of any deviation in the functioning of the instrument from one assay to the next. 9.3.5. Line-reagent and reaction-mixture caIcuIations Details about the reagents pumped in each line, their final required concentrations and their concentrations in stock reagents are entered here. The computer prints out the dilution of stock solution required to give the correct final concentration in the reaction mixture, taking into account the observed line volumes calculated in the previous section. Thus, if one of the lines begins to pump poorly, the dilution of stock reagent would be adjusted so as to give the correct final concentration. 9.3.6. Stock soIut ions Molecular weights, required volumes and concentrations of each stock solution are entered here and a weighing sheet is printed showing the required weight for each solution with a space for entering the weight of the vessel. These weighing sheets may be accumulated and used repeatedly. 9.3.7. Gradient calibration The reading and blank with undiluted marker are enteredhere, followed by the gradient-calibration readings as the marker solution is pumped

GENERALIZED SYSTEM

151

into the gradient-making system. The gradient dilution factor is calculated for each reading and the results are printed as a table and a graph. 5.3.8. Assay conditions Comments about the assay, such as temperature and sampling rate are conveniently entered here. If one is preparing a detailed report or paper, it is convenient to enter textual material here, so that the final computer output will contain text, tables and figures. 5.3.9. Protein concentration Three possible situations are dealt with. In the first (‘CONSTANT’) no gradient-making system is used and the protein concentration is the same in all assays. This is used in simpler M.E.A. systems or during enzyme characterization studies. The protein concentration in the reaction mixture is then calculated from the protein concentration in the enzyme line and the fractional line volume of that line. In the second situation (‘VARIABLE’) the protein concentration in each sample is different, as in enzyme sample determination. In this case the protein concentration in the reaction is calculated for each sample from the initial protein concentration in the sample cup. (A system is under development in which the protein concentration will be determined by an on-line protein analyzer and the data froni this fed in at the same time as the enzyme results.) In the third situation (‘GRADIENT’) the protein concentration in the reaction mixture varies because of the action of the gradient-making system. This is the case in most recently developed M.E.A. systems. The protein concentration is then calculated from the initial protein concentration in the stock solution of enzyme dripping into the gradient-mixing vessel, and the gradient dilution factor for each reading calculated previously. The various protein concentrations are printed as tables, and where appropriate, as graphs of protein concentration against reading number. 5.3.10. Sample pattern Details of the samples are entered here including the concentration of Subjecr index n. 218

152

AUTOMATED ENZYME ASSAYS

any components in the samples if known. Thus if the sample pattern consists of substrates for M.E.A., initial substrate concentrations are entered. The sample pattern is printed out with initial and final concentrations.

9.3.1 I . Results These are calculated for a variety of different situations. Allowance is made for the three types of protein concentration calculations mentioned in § 9.3.9. In addition, calculations are made in a slightly different way if the protein concentration is not entered. Not all assays use the sampler module and these also have a slightly different way of calculation and print-out. In all cases the results are calculated for each reading in terms of pmoles/ml of reaction mixture and pmoles/min. Where the protein concentration is known they are also calculated as pmoles/mg protein/min. All results are printed as a table and appropriate comments may be inserted in the legend. For each of the above situations, the instruction ‘GRAPH’ allows a plot of both pmoles/ml and pmoles/mg protein/min against reading number. Again, comments of any length may be inserted in the legend. The graphs are plotted as percent of maximum value in the given set of readings, and at the same time suitable scale divisions on the ordinate scale are inserted in absolute units. 9.3.12. Computer sub-routines The program given in appendix IV includes two sub-routines. The first (‘COMMENT’) allows one to print out any material of any length either to check the data before running on the computer, or simply for record purposes (‘LISTDATA’ entry). The second subroutine (‘PLOT’) will produce a graph of any series of values against reading number as described in the previous section.

9.4. Example of the use of the above system Details of a typical assay will now be given; it is a multiple-enzyme assay for three dehydrogenases, following the reduction of ferricyanide

GENERALIZED SYSTEM

153

at 420 nm. The reaction mixture required is 0.4 mM K,Fe(CN),, 0.05 M K phosphate p H 7.4, and any of the three substrates 2.0 mM Na L( +)-lactate, Na D(-)-lactate or Na succinate. The reaction takes place at 37 "C and the protein concentration should be in the range 0.1-2.0 mg/ml and the time of incubation should be about 5-10 min. The instrument is first thoroughly washed with 1 N NaOH, water, 1% Triton-X-100 and water, and the flow cell is disconnected from the heating bath. A series of solutions of increasing concentration of potassium ferricyanide are then pumped directly through the flow cell and the reading and blank recorded. The flow cell is then re-connected and marker (0.1 M potassium ferricyanide) is introduced into the enzyme line for about 5 sec, with all other lines in water. When the marker enters the jacketed mixing coil before the heating bath, a shutter is momentarily placed in front of the sample beam of light in

C h a r t divisions

Fig. 9.9. Method of calibrating gradients. 2 mM potassium ferricyanide is placed in the gradient-making system and water is placed in odd-numbered cups in the sampler. The even-numbered cups are left empty. The ferricyanide is diluted as the water is sampled and the gradient dilution curve is formed by joining the troughs (dashed line). Numbers on the trace refer to the cup number. (From Roodyn 1967b.) Subject indes P. 218

154

AUTOMATED ENZYME ASSAYS

the colorimeter. This causes the pen recorder to mark a clear line on the chart. When the marker enters the flow cell a rapid upward trace on the recorder marks the end of the incubation. The distance between these lines is measured and the chart speed is determined by measuring the distance between two lines marked 5 min apart. (These measurements are usually repeated three times and the average values used.) The line volumes are then determined by placing each line in turn in 1 mM potassium ferricyanide for 5 min, followed by 5 min in water. The base-line and reading for each line is recorded and the total volume of fluid pumped by the analyzer is measured by collecting all the output from the waste lines for a known time in a measuring cylinder. (Mr. G. K. Jowett in our laboratory has recently suggested a simpler method of line calibration : Each line in turn is connected to a burette on the input side of the pump, and the time taken to empty a given volume into the pump is recorded.) The gradient-making system is calibrated by placing 25 ml distilled water in the gradient-making vessel (mixer 1) and 2 mM potassium ferricyanide in the stock vessel (mixer 2). Water is placed in all sample cups and the sample-wash line is disconnected. As soon as the ferricyanide starts to drip into the gradient-making vessel, the sampler is started from the wash position, at 40 samples/hr. An example of the resultant trace is shown in fig. 9.9. (For clarity, the calibration was performed with water in alternate cups.) The trough value for each cup is measured, as well as the initial and final blanks. When the gradient is completed, 2 mM ferricyanide is run directly into the enzyme line, and the reading and blank recorded. The above data about molar conversion factors, incubation times, chart speed, line volumes and gradient calibration are entered into the computer with details of the composition of the reaction mixture and the molecular weights of the solutes for the stock solutions. The output is shown as follows: molar conversion factors and incubation time in table 9.1, line volume calculations in table 9.2, stock solutions in table 9.3, line reagents in table 9.4, and gradient dilution factors in table 9.5. and fig. 9.10. From this information, the line reagents and sample table are prepared and a suitable dilution of enzyme is placed in the stock mixing vessel at 0 "C.The assay is then started by connect-

155

GENERALIZED SYSTEM

TABLE9.1 C

A

L

I

B

R

A

T

I

O

N

MOLAR C O N V E R S I O N F A C T O R C A L C U L A T I O N CHROHOPHORE CONCENTRATION IUMOLESIMLI

READING

BLANK

0.100 0.200 0.300

0.144

0.019

0.271 0.396 0.520 0.643 0.768 0.065 0.955

0.019 0 .019 . . 0.019 0.020

0.400 0,100 0.bOO

0.700 0.000

A R E A D I N G OF

MOLAR C O N V E R S I O N FACTORIUHOLESIML THAT G I V E A R E A D I N G O F 110

0.800 0.7Pl 0.796 0.798 0.803 0.802

0.020

0.020 0.021

1.0 IS E O U I V A L E N T I0

YITH b P I T Y L E N G T H O F 1 5 . 0 HM.

CHROHOPHORE C O N C E N T R A T I O N U P T O T H E CHROMOPHORE IS I( F E R R I C Y A N I D E

0.828 0.057

0 . 8 1 O U H O L E S / M L A T 420,O MU THE READINGS I R E PROPORTIONAL 0.800 UMOLES/ML.

TO

S T b U D A R O S O L U T I O N S PUMPEO O I R E C T L Y I N T O T H E F L O W C E L L OF T H E C O L O R I M E I E R . I N C U B A I I O Y T I M E AN0 CHART SPEEO C A L C U L A T I O N T H E C H A R T YAS R U N FOR 6 1 0 P I I N S . A N D M O V E 0 4.5 CMS. FOR I N C U B A T I O N T I M E MEASUREPINT Y E R E 4 . 0 CM. APART T H E C H A R T S P E E D Y A S 0.753 CHIWIN. AND THE INCUBATION

MARKS

TM IE

5 , s HIN.

V A R I A B L E S P E E D PUMP S E T A T P O S I T I O N 7 0

TABLE 9.2

L I N E VOLUME C A L C U L A T I O N LINE NUMBER

1

**.

**.

LINE COLOUR YYITE

YbRNlNG L I N E 2 GREEN 3 YHITE YARNING LINE 4 YELLOY

CALIBRbTIONREADING

BLANK

0.193

0.029

FRACTIONAL LINE VOLUME

0.113

LINE

D I L U T I ON FACTOR

...

d151

1 H A 5 MORE T H A N 1 0 P E R C E N T P U I l P l N G ERROR 0.4P3 0.029 0.153 2.51 0.189 0.029 0.149 6.70

3 HAS MORE T H A N 1 0 P E R C E N T L U M P I N G ERROR *a. 0.313 0.029 0.265 3.77

OBSERVED LINE VOLUfIE I*L/MINl

EXPECTED LINE VOLUPIE IMLIMINI

0.b2

0.55

1.76

1.84

0,)s

0.bL

i.08

O B S E R V E D TOTAL L I N E VOLUME I S 4.06 M L I H I N . E X P E C T E D T O T A L L I N E VOLUME O B S E R V E D V A L U E IS 1 0 0 . 2 P E R CENT OF E X P E C T E D

1.11

IS

LINE ACcUnAcv I P E R CENT OF T Y E O R V I

112.9

95.5

110.2

96.9

4.05MLIMIN.

T H E O R E T I C A L V A L U E S C A L C U L A T E D FROM H I K E R ' S S P E C I F I C A T I O N S , P U M P S P E E D O F S T A N D A R D T E C H N I C O N PUMP A N 0 O B S E I I V E O SPEEO O F V A R I A B L E S P E E O PUMP. I T Y E P U M P I N G ERROR I S P R O B A B L Y B E C A U S E T H E PUMP IS N O T R U N AT I H E S T A N D A R D I.CC" =-EL".

I H E M A N I F O L D U S E D Y A S AS F O L L O Y S L I N E NO.

COLOUR

COMPONENT I N L I N E

01 02

YHlTE S U E S T R A T E S t I N S I M P L E R I I MODULE GREEN BUFFER 0 GAS BLUE 03 YHlTE CO-FACTOR 04 'IELLOY ENZ'IME,FROM O R A D l e N T M A K I N P SYSTEM. YHlTE OR O R I D I E N T L I N E n T O G R A D I E N T M A K l N O SVSTEM. Y PURPLE BLACK YASTE L I N E T H E SAMPLER WAS O P E R A T E D A T I 0 S A L P L E S P E R H O U R , Y I T H WATER I N THE MASH V E S S E L . T H E H E A T I N G BATH Y A 5 A T 37 c.

Subject index P. 218

156

AUTOMATED ENZYME ASSAYS

TABLE9.3 PREPARATlON OF SOLUllONS COMPONENT OF STOCK SOLUllON

MOLECULAR Y E I O H T OF COMPONENT

W fERRlCVANlOE

CONCENTRATION OF COMPONENT I N STOCK SOLUTION

0.010

329.3

UNITS OF CONCN.

VOLUME Or STOCU S O L U l I O N REPUlREO IML.)

MOLAR

2001000

YEIOYT OF CONPONENT (OM)

YEIOHT Of VESSEL YEIOHT OF COMPOhCNT

_-__--_._ 0.65860

SUM

L l r ) L A C l A l E - C I SALT

296.2

0.050

2501000

MOLAR

Y E l O H l OF VESSEL YEIQHT OF COMPONENT SUM

Dl-)LACTATE-CA

SALT

290.2

0.050

MOLAR

210,000

_____ -__3.70210

YElOHT OF VESSEL Y E l Q H l Of COMPONENT

3.62750

SUM YElQHT Of

0.050

118.1

SUCCINIC ACID

250,100

MOLAR

VESSEL

YEIOHT OF COMPONINT

1.4?625 _____-___

SUM

1Sb.l

KH2POI

1.000

5000.000

MOLAR

WEIPHT O f VESSEL YEIOHT Of COMPONENI

-------.6110.50000

SUM

E l LACTATES CONVERTED TO NA S A L T S BY TREATMENT Y l T H NASPO4. SUCClNlC ACID NEUTRALlZED Y I T H NAOY. KH2PO4 ADJUSTED T O PH 7.4 Y l l H KOH.

TABLE 9.4 L I N E REAQENT AND REACTION MIXTURE C A L C U L A l t O N LINE NUMBER

COMPONKNT I N REACT.IDN MIXTURE

1 NA Ll.) LAClAlE I NA D ( - 1 LACTATE 1

2 3

NA SUCCINITE K PHOSPHATE PW 7,4 KIFECN6

CONCENTRATION OF COMPONENT I N 1l)LINE i2)REACTION 131STOCK MIXTURE SL)LUTION 13.07S 2.000 10.000 2.000 50.000 13.073

REAOENT 15.073 0.116 2.610

z.000 0,050

0.400

1o.ooo

1.000 10.000

UNITS

OF

CONCN.

MlLLIM MILLIM MlLLIM MOLAR

MILLIM

TABLE 9.5 ORADIENT CALIBRATION READIN0 NUMBER

REIDlNQ

TO PREPARE L I N E REAOENT MAKE I A I M L . OF STOCK UPTO IA) (0)

QRADlEHT DILUTION FACTOR

65.37 65.31 65.37

577.59 134.00

250.00 250.00 250.00

5000.00 ¶OO.OO

151

GENERALIZED SYSTEM

.

0

-It c

e a t

.

. .

D

.

mOt

.

D

.

C t

O

O

. .

t

.

O

.

In.

.. .. ... ... .. ... .. .. . ... ... . ... .. .. . ... . ... .. .. . .. ... . ...

..

. . ... .. ... .. . ... .. -. ... . ... .. . .... . ... .. ... .. . .... ... . ...

C

.

v +

L D

C

n

" W

C O

a Y a

o

z . i

O

.

*)+

O

.

N t

O

.

d l

O t

.

z D

.

i

a

...

...

.. .. ... ... .. ... . ... . ... . .. .. ... ... ... ... .. .. .. ... ... ... .. .. .. ... ... .. .. .. .. ... ..- ... . . . ... ... ... .. .. .. ... ... ... . . . ... ... ... .. .. .. ... ... ... ... ... ... . . . ... ... ... ... ... .. ._. ... .. *. .. .. .. .. . . . .... .... ... . . .. .... .... ... . . .. ... ... ... . . . ... ... ... . . . ... ... ... .. .. .. .. .. .. ... ... ... .. .. ..

.. ... . ... .. . ... .. ... ..

.. ... . ... .. . ... . ... ...

t

... ... ... . . . ... ... ... . . . ... ... ... . . . .. .. .. ... ... ... . . . .. .. .. ... ... ... .. .. .. .. .. .. .. .. .. ... ... ... .. .. .. . . . .. .. ... .. .. .. .. .. .. _ .. ... ... ... .. .. .. .. .. .. ... ... ... .. .. ... ... .. .. ... ... . . . ..... ..... ..... . . .

.

.. . ... .. ... ... ... .. ... ... .. .. . ... .. .._ . ... ... ... . ... .

.

-

. ... .. .

.. .

... .. ...

.. .. .. .. .. . .. .. .. ... .. .. .. .. .. .. .. ... ... ... .. .. .. . . . .. . . .. .. .. .. ... ... . . . . .~. . .. .. .. ... .. .. . . . .. .. ... ... ... .. .. .. .. .. .. .. .. .. .. ... ... ... . .. .. .. ... ... ... .... .. .. .. .. .. .. . . . . . . . . . . . .. .. .. .. .. ... ... ... ... ... ... ... *. .. .. .. . . . . . . ... ... ... ... ... ... ... .. . . . . . .. .. .. .. .. ... ... -... ... ... ... ... ... ... ... ... .. .. .. .. _ . .. . . . ... ... ... ... ... .._ .. . ... ... ... ... ... .. .. ._ . .. .. .. . ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . .. .. .. .. .. .. .. . ... ... ... ... ... ... ... .... . . . . . . . .. .. .. .. .. .. .. ...

.. . ... .. .

.. . ...

...

f

. ...

. ... ... ... .. ...

. .. ... . ... ... ... ..

. .. ... . ... ... ... ..

. ... ... .

.

. ... ... .

.

. ... ... .

. ... ... .

. . . .

. ... . . . .. .. .. ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. . . . . . . . . ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. . . . . . . . . .. .. .. .. .. .. .. ..

u s o r .

E

C

D .

O i

r r

t (

-

N *

.. +

. ..... . - . ... . . . ...

...

.. ... ... .. .. .. ..

... . . . . .. .. .. ... ... . . . . ... ... ... ... .. .. .. .. .. .. .. .. . . . . ... ... ... ... .. .. .. .. .. .. .. ..

...

n

.. . ... .. ... ... ... .. ... ... .. .. . ... .. ... . ... ... ... .. ...

D r

(

. ... .. ... ... . ... .

O r

O l

.. .. .

. .

. .

. ... * . .. .. .-. * ..

.. ... . ...

.

. ... .. ...

.

. . .._ .. .. .. ... ..

O C E - 4 . 4

d P

..

..

..

.

. *

. ... ... . . * ... .. .. . t .. ... . .. . t ..

.. .. ... .... + ... .-.. * ....

... ... ... . . . . . . .. ... ... ... ... . . . . _ .. .. . .. _.. .. .. .. .. .. .. .. .. .. ... ... ... . . . . . . . f

L

+

..

O

.

D

.

0

m

-

.

0

.

Z

.

O

z

0

I

. . .

>

m

.

.

m -

-

. .

0

U

.

w m

. . ..

I

. -

> m Y

.

Z

-

x

+

... .. N O

Fig . 9.10. Plot of gradient dilution factor against reading number (computer output).

Subjecr index p . 218

158

AUTOMATED ENZYME ASSAYS

ing the appropriate reagents to the various lines, checking that the base-line is steady and starting the sampler as soon as the stock enzyme starts to drip into 25 ml of diluent (0.3 M mannitol) in the gradient-mixing vessel. The readings are taken from the steady-state values with each peak. The base-line rises during the assay because of absorption at 420 nm caused by the steadily rising enzyme concentration. If this rise appears to be linear, it is possible to use only initial and final blank values, using the computer to calculate the blanks for each sample by linear interpolation. However, it is more precise to measure the true blanks by drawing a line connecting all the blank samples in the assay and dropping a perpendicular line from the steady state portion of each peak to this base-line in order to calculate the true blank for each reading. The method of doing this is shown diagrammatically in fig. 9.1 l . The following information is then entered into the computer: the initial protein concentration in the stock mixing vessel, the nature and initial concentration of substrates in the sampler cups, the readings and blanks for each cup, and comments on the experiment where appropriate. The output is as follows: protein concentrations for each reading (table 9.6 and fig. 9.12), details of the sample pattern and substrate concentrations in the reaction mixture (table 9.7), the actual results of the enzyme assays (table 9.8), and graphs of enzyme activity in pmoles/ ml and pmoles/mg protein/min against reading number (figs. 9.13 and 9.14). This example has been given in detail in order to show the flexibility of the system, and also the wealth of data from instrument calibrations and readings that may be handled by the computer. The close interaction between computer program, autoanalyzer and human operator has its difficulties. In the above system, the data were punched manually on IBM cards and batch-processed on a large central computer. This lead to delays that were particularly annoying when an error had been made in input. Ideally, the analyzer should be on-line to a large computer capable of handling a program of the size shown in appendix IV. However, the recent expansion in small, relatively cheap, on-line computers may provide an alternative, more practical scheme. In

GENERALIZED SYSTEM

159

Fig. 9.11. Method of calculating blanks. (a) With blanks that increase or decrease linearly, linear interpolation is carried out, using the initial (I) and final (F) blank values. (b) With a non-linear change in the blank, blank values are obtained for each peak by dropping a vertical line (arrows) to the line connecting the various blanks interposed between the readings (dotted line). B : blank; R: reading. (The traces are hypothetical.) TABLE9.6 P R O T E I N CONCENTRATlPNS T H E E N Z Y M E H l S e N Z Y M E SAMPLE 1 6

UITH I N I T I A L P R O ~ E I NC O N C E N l R A T l O N OF

1.050MG1ML

P R O T E I N CONCENTRATION I N REACTION M I X T U R E DURING GRADIENT REIDING NUMBER

GRADIENT O I L U l I O N FACTOR 0.062 0.098

0.136

P R O T E I N CONCN. I N REACTION MIXTURE 0.017 0.027 0.030

6 7

0.173 0.209 0.243 0.278

0.040 0.050 0.068 0.077

0

0.316

o.ow

0.345 0.379 0.414 0.446 0.403 0.5011 0.542 0.573 0,604 0.635 0.660 0.702

0.096 0,106 0.115 0.124 0.134

1

2

3 4 5

9

10 11 12

13 14

15 16 17 10 19 20

O.l*l

0,151 0.159 0.160 0.177

0.104 0.195

Subjecr index P. 118

160

AUTOMATED ENZYME ASSAYS

TABLE 9.7 SAMPLE PATTERN SAMPLE NUMBER

1 2 3 4 5 6 7

1 9 10

11

SAMPLE COMPONENT NUMBER

1 2

3 4

1 2

3 4

1 2

3

12 13 14

4

15 16 17

3 4

10 19 20

1 2

1 2

3 4

NA LI.) LACTATE NA 01-) L A C T A T E NA SUCCINATE YATER NA L l * I LACTATE NA 0 1 - ) L A C T A T E NA S U C C I N A T E YATER N A LI.1 LACTATE NA 0 1 - 1 L A C T A T E NA S U C C I N A T E HATER NA L l f ) L A C T A T E NA 0 1 - 1 L A C T A T E NA S U C C I N A T E YATER NA LI.) LACTATE NA 01.) L A C T A T E NA S U C C J N A T E HATER

MET1100 FOR L'OADINO S A M P L E T A B L E WBSTRAiE

C O N C E N T R A T J O N OF S A M P L E COMPONENT I N

S A M P L E COMPONENT

LINE

REACTION

13.100 13.100 13.100 0.000

2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000 2.004 2.004 2.00* 0.00.0 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000

13.100

13.100 13.100 0.000 13.100 13.100 13.100 0.000 13.100 13.100

13.100

0.000 13.100

13.100

13.100

0.000

UNITS OF CONCN.

MIXTURE

MiLLin MILLIR MILLIR MlLLlR MILLIN MILLIU MlLLlI MILLIM MILLII MILLIM MILLIM MILLII MILLIM MILLII MlLLlM

-

CUPS

0 1 05 0 9 1 3 1 7 2 1 NA L l * I LACTATE N A DI-I L A C T A T E 0 2 06 1 0 1 4 1 0 2 2 NA SUCCJNATE 03 0 7 11 1 5 1 9 2 3 YATER 0 4 08 1 2 16 20 2 4 STOPS AT CUPS 2 0 AND 40 THIS A L L O Y S FOR THO ASSAYS P E R P L A T E

25 26 27 28

2 9 33 37 30 34 31 3 1 35 3 9 5 2 36 4 0

TABLE 9.8 PATE 1 5 / 5 / 6 9 IXPT. 01 RESULTS

OF E N Z Y M E ASSAYS

l E A D l N Q SAMPLE S A M P L E COMPONENT NUMBER COMPONENT NUMBIR

f

1

2. 3

2 3

4 3

4 1

6 7

2 3

a

6 ta

.

1

2

11

3

13

4 L 2

12

".,En

ii'Ci.1

J

LACTATE NA 01-) L A C T A T E NA SUCCINATE HATER NA L l t l L A C T A T E NA Dl-I L A C T A T E NA SUCCINATE

4

HATER

14 15 16 17

3 4

18

2

19 20

NA L l r ) LACTATE NA 01-1 L A C T A T E NA S U C C I N A T E YATER NA L l t I L A C T A T E NA DI-I L A C T A T E NA SUCCINATE YATEll NA L l r ) LACTATE NA D l - ) LACTATE HA S U C C I N A T E

1

CONCN, OF UNITS SAMPLE OF COMPONENT CONCN. I N REACTION MIXTURE 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000

MlLLlM MILLIM MILLIM

READING BLANK

01461

0.479

ENZYME A C T I V I T Y UMOLESlML UMOLESIML UMOLEIIMP. IMIN PROTElN/MlN

-0.OlY

-0.003 -0.009

-

-0.001 -0.000

-

-0.010 -0.025 -0.002 -0.000

MILLIM MILLIM MlLLIM

-0.016

MILL111 MILLIH MILLIM

-0.034 -0.002 0.000 -0.021 -0.044 -0.002 -0.000 -0.027 -0.056 -0.002

-

MlLLlM MILLIM MILLIM

-

MILL111 MILLIM MlLLIM

-

T A E L E 95 ENZYME PATTERN I N D L U C O S l REPRESSED CELLS. ( V A L U E S I R E N E O A T I V E V E C A U S E THE D I S A P P E A R A N C E OF K F E R l C V A N J D E W E A S U R E U I N THE A S S A Y . I

0.000

IS

-0.07

-0.33b

-0.020 -0.009 -0.168 -0.340 -0.019 -0.00Y -0.168 -0.325

-0.011

0.002 -0.158 -0.314 -0.016

-o.ooa

-0.16L -0.316 -0.008 0.000

161

GENERALIZED SYSTEM

.. ... ..

... ... ... ... ... .. . .. ... ... ... ... ... ... ... ... ... ... ... ... . + . . . . . . . . . . . . . . . . . . . ... ... ... ... ... ... .. ... ... ... ... ... ... ... ... ... ... ... ... . .. .. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. . . . . . . . .: .: .: .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. , .. .. .. ... ... .. . .. . .. ... ... ... ... ... ... ... ... ... ... ... ... . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . .. .. .. .. .. .. .. . , .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . ... ... ... ... ... .. . .. ... ... ... ... ... ... ... ... ... ... .. ... ... . . .. .. .. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. .. .. . ... ... ... ... ... ... ... . .. . ... ... ... ... ... ... ... ... ... ... ... .. . . . . . . . . . . . . . . . . . . . . . ... ... ... ... ... ..... ... ... .. . ... ... ... . ... ... ... ... ... ... ... . .. .. .. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . .. . .. .- .> .: . :. :. :. :. :. : : : : : : : : : .. .. .. .. .. .. . . . .. .. .. .. ... ... ... ... ... ... ...... . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . . . . . . . . . . . . . . . . . . . n . . . . . . . . . . . . . . . . . . . . . . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . z . .: . : . : . : . : . :. :. :. :. :. :. .: .: .: .: .: .: .: . : : . .. ... ... ... ... ... ... ... . .. . ... ... ... ... ... ... ... ... ... ... ... .. 0 ,-$* D

-

0

P ,

0

I

0

I

O I

I..

p. u m

x

2 3

" z

.

I

0

w

p.

I-

-"

Y)

z

<

<

== Ex

<

x

L. 0

O

.

.

0 .

.

.

I

*

0

-

I

-

0 0

I. 0

-0 z Y)

I-

m z

0 0

= w El z 1 0

p. 0

VI -1

A " YI

I 0 . 0 LL

t

0

L A

-

0

z

-

N

n

t

0

0

-

-

O

D

d N - - -

n t - - ,

& 3 O L - $ d - - r

O O I N

I

Fig. 9.12. Plot of protein concentration against reading number (computer output).

Subject index P. 218

162

AUTOMATED ENZYME ASSAYS

0

0 .

.. ... ..

... .. ... ..

O I

-

.. ... ..

... ... .

... ... .

... ... .

... ... .

... ... .

... ... .

.

N

W

t

R

c

f

i

O

t

... ... .

... ... .

... ... .

.. ..

.. ..

.. ..

.. ..

O

o -

4

-

..

yiii;;

.. ..

.. .. .. . . . . .. . . . .... .... .... .... .... .... ..... ...w . ; .- .: .. .. .. .. .. .. .. .. ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. +

-

. . ... ... .. .

. . * .. :. :. : .: / ; t :. : . : . ... ... .. .. .. . ... ... ... ... ... ,

. . . . . . . . . . .

0 .

0 7

-

.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. ... ... ... ... ... ... ... ... .- / " : ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . .. . .. . ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..

0 4. D

4 4

.. ..

.. ..

.. ..

.. ..

.. ..

.. ..

. -

- ... ' ***

z

"

. . . . . . . *. * . .:. :. .... ..... .. ._... .._.. _ _ .. .. .. .. .. .. .. .. .. .. .. .. .. .. -

4

n d

r -

m d

-

o 4

h d

c d

d

a

o

d

N

*

m . c

.

n -

.. ..

O

Fig. 9.13. Plot of enzyme activity in pmoles/ml against reading number (computer output). For clarity, lines connecting the asterisks have been entered by hand. L: L(+)-lactate dehydrogenase; D : D(-)-lactate dehydrogenase; S: succinate dehydrogenase; and W: water blanks.

163

GENERALIZED SYSTEM

. . . . . . :. ; . .: . -;\o. .

0

. o = I .

; .;.

.: :. :. : . : .: .: : : : \ ; 0

D

t

0

O I

O

C .

.. . .. ..

.

.. .. .. .. . ... .. .. ... . ... . ... .. .. ... ..

0 LL

O l

.. .. . ... . ... .. ..

z

0

r

. :. .i

. :. ...

. :. ...

. :. . ..

. . :. :. . .. . ..

.. ... ... ... ... ... ... ... . . . . . .. .. .- . .. . . ... . ... .. ... ... ... ... .. . .. . .. ... ... ... ... ... ... ... ... ... ... ... . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..

. ... .. .. .. ... .. ... .. .. ... . ... .. .. ... . ... ..

:-:*.:. . . . .. .. .. . ... .. ... . ... .. .. .. .. ... -....

. : . .

.. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . . . . . . . . . . . . . . . . . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . .. .. .. .. .. .. .. .. .. . ... ... ... . --. ... ... ... ... ... ... ... ... ... ... ... ... ... .. . . . . . . . . . . . . . . . . . . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .- .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . :L: . . .: .: .: .: .: .: .: .: .: ... .... .... .... .... .... ... .... .... .... .... .... ... .... .... .... ....... . . . . . . . . . . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. . . . . . . . .. .. .. .. .. .. .. .. .. . ... ... . .. . ... ... ... ... ... ... ... .... ... ... ... ... ... .. . . . ._ . . . .. .. .. . . .. .. .. .. . . . . . .. .. .. . . + .. .. .. .. .. . . . . . . . . . .. .. .. .. .. .. .. .. . t

I

m -. W m

.-=

.. ... .. o .. o * ... .. .. . . . . . . . . . . . . . . . . . . - - . .. . . . . . . . . . . . . - . ... ... ... ... ... ... ... ................ . .. . .. . .. ... ....... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . * u .. .. .. .. .. . . . . . . . .. .. .. .. .. .. .. .. .. .. .. . z ... ... ... ... .. . .. ... ... ... ... ... ... ... ... ... .. ... ... _ . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. .. .. . -> .. .. .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. . w ... ... ... ... ... ... ... ... i s ;.: .: :. ... ... ... ... ... ... . * .. .- . . .. .. .. . . . . .. ... ... ... .. ... ... ... ; :-?--?=. . *: + .. .. -. .. . . . .... .... .... .... .... .... .... .... .... ..... ... .... .... .... .... .... .... .... ... .. .. .. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. .. . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 2 . 2 c: :: :: :2 2 : s z

.-

. .

D

.

<

:. fT.

..

. -

- z . u

D

O

I

VI -

El

L

0

*

~

O

h

~

. < u . .

1

3

n

c

. V I Z

A

N

D c

.

<

l

2

. . W aa

O

V

..

Z3

.. ".

.

r

0 . P &

.. . . . ,r - n .

.

O

. .

. I

-

W

2

0 ;

3

. - I .0

. <

..- I a -

-

m

u

~

Fig. 9.14.. Plot of enzyme activity inpmoles/mg protein/min against reading number (computer output). Lines and symbols as in fig. 9.13.

Sehiecr index P. 218

164

AUTOMATED ENZYME ASSAYS

this, the small computer handles the immediate output from the analyzer and issues warnings about the operation of the instrument during the actual assay. The small computer may then perform part of the data-processing and produce a physical record (e.g., paper tape) which can then be processed on the large computer in more detail by more extensive and sophisticated programs. However, provided that the input of data is very carefully checked in order to avoid time-consuming errors, the system that I have used should be helpful to anyone with an automated assay system and with access to a computer centre. Since the number of such people is increasing steadily, it is hoped that the generalized approach will be of use perhaps even to analysts who are not concerned directly with enzyme assays. Indeed many of the problems of instrument calibration, preparation of reagents and calculations of readings and blanks arise in all automated assays so that the above treatment may be widely applicable.

Conclusions and future prospects

We have seen how the various processes involved in the assay of an enzyme from the preparation of reagents to the final tabulation and plotting of results may be automated. Many possible systems exist and the choice will be determined partly by the problem under study but, I suspect, primarily by the availability of funds. It does not always follow that the most fully automated systems are the most suitable. The problem is one of combining automation with what I might call ‘operator accessibility’ to add yet one more term of jargon! If a complex and expensive system has been elaborated it may become difficult to make minor adjustments without upsetting the flow of work. A situation similar t o that in computer centres may arise, in which in view of the large capital cost and staffing and maintenance charges, it will become imperative to operate the system almost continuously. In fact, the future may well see the establishment of ‘analytical centres’ serving several laboratories with automated analyses. It may be possible to incorporate flexibility into such ‘multi-access’ centres by constructing the instruments from generalized analytical modules of the sort described in the previous chapter. The user may supply the stock solutions, the samples and a program indicating the sequence of operations to be performed with them. The centre may also have certain standard assays, analogous to the ‘library’ sub-routines of the computer centre. Whatever the exact line of future development, there is no doubt that a wide range of automated systems of varying complexity will be 165

Subiecr index P. 218

166

AUTOMATED ENZYME ASSAYS

used to a great extent in the near future. In routine laboratories, whether clinical or industrial, the advantages that accrue will be considerable, although they may have certain social results in their effect on the type of staff that is employed. I n some ways, more skilled staff will be required, particularly for maintenance of complex equipment and data-processing. However, at the same time, routine ‘analysts’ may well find themselves in danger of replacement by less skilled ‘analyzer operators’. It is difficult to assess the impact of full enzyme automation on pure research laboratories. Because of the nature of basic research, with its halting and unequal rate of progress and its frequent encounter with the unexpected and the irritating, it is difficult to set up ‘routine’ analytical systems that are fully employed. For example, in the past it would have taken about a month to perform preliminary characterization studies on an enzyme, and possibly 1-6 months to achieve a satisfactory purification. During that time, various theories could be proposed and then disproved, literature surveys could be conducted, seminars and discussions used to adjust the lines of work. However, with automated methods, these studies could be completed in a matter of weeks, if not days. Attitudes of scientists have evolved in an atmosphere based on the rate of performance of manual operations. An enzymologist would hesitate to design a series of experiments in which detailed kinetic studies were performed on twenty different enzymes in one week. However, such is the potentiality of modern methods, we can now envisage a situation in which a single research worker with access to fully automated systems, including extensive data- and literaturehandling capabilities, could well have the scientific productivity of an entire department! Let us assume that he has a four-channel sensor operating at 60 samples per hour, i.e., giving 240 new values per hour. If he operates this system for five days a week and eight hours per day, he will produce about 30,000 readings and blanks a month. If we assume that only 10% of this has any scientific value (and it may all have value), and we estimate that about 30 values are needed to produce a table or graph, his output per month would be 100 tables or graphs, enough for ten medium-sized papers or reports. Extending the fantasy

161

CONCLUSIONS AND FUTURE PROSPECTS

further, a small group of five workers each equipped in this way could produce 600 detailed papers or reports per annum even if they rejected 9/10 of their work! It is clear that conventional forms of scientific reporting, in particular the standard paper to a journal, would not be adequate to cope with the flood of information that fully automated systems could generate. New methods will have to be developed to disseminate this information, with the computer playing a central role. Probably only a part of the information will be made available to other scientists, or even written in formal reports. There will be an ever increasing accumulation of unpublished material of varying degrees of intelligibility. A symptom of this is the increasing appearance in the biochemical literature of very large papers that are difficult to assess in a reasonable time by the general reader. The new techniques may well produce a crisis in the scientific literature. We will only be rescued from it by imaginative developments of methods of handling scientific output and of bringing relevant information to those who need it without overwhelming others with a flood of ‘literature’. It would indeed be tragic if the great advantages that could result from the general application of the automated processes would come to nothing because of a breakdown in our system of scientific publication and exchange.

Subjecr index

8.

218

APPENDIX I

Published automated enzyme assays

In some of the references in this appendix full details are given including preparation and dilution of reagents. In others the systems are presented in a more generalized way. In view of the absence of a comprehensive index of automated assays, both types of paper have been included. In some cases, the assay has been designed to estimate a substrate, rather than the enzyme. In such cases the substrate is given in brackets after the enzyme (e.g. Glucose oxidase (glucose) Hill & Kessler 1961). Enzyme assayed

References

Abou-Donia and Menzel 1967; Groff et al. 1966; Serrone et al. 1965 Acetyl thiocholinesterase Humison and Wright 1967 Acid phosphatase Bodansky and Schwartz 1961; Chersi et al. 1967; Green et al. 1966, 1967; Klein and Auerbach 1966; Klein et al. 1965, 1966; Leighton et al. 1968; Schuel and Anderson 1964; Stein and Lewis 1966; Tappel 1964 Alcohol dehydrogenase Roodyn 1965b; Schwartz et al. 1961 Alcohol dehydrogenase (alcohol) Syed 1967, 1968 Aldolase Kaldor and Schiavone (1968) Alkaline phosphatase Axelsson et al. 1965; Birkett et al. 1967; Coleman and Stroje 1965; Comfort and Campbell 1966; Cooke and Patston 1961; Everard and Seymour 1963; Fishman and Gosh 1967; Green et al. 1966; Hill et al. 1968; Horne et Acetyl cholinesterase

168

PUBLICATIONS

Enzyme assayed

Amidases Amido transferase Amino acid decarboxylase L Amino acid oxidase (L amino acids) D Amino acid oxidase Amylase

Aromatic acid decarboxylase Aromatic ketolenol tautomerase ATPase ATPase (Na+, K+ activated) Catalase Cathepsin Ceruloplasmin Cholinesterase Cholinesterase (organic pesticides) Citrate cleavage enzyme Creatine phosphokinase Cytochrome oxidase DOPA decarboxylase Ferricyanide reductases Ficin Formylase Fructose aldolase Galactosidase p-Galactosidase o-Galactosidase o-Galactosidase p-Galac tosidase

169

References al. 1968; Hviid 1967; Keay and Trew 1964; Klein and Kaufman 1967; Marrack and Hall 1965; Marsh et al. 1959; Morgenstern et al. 1965c,d; Roos 1965,1966; Sterlinget al. 1964; Tietz and Green I964 Lenard et al. 1965 Pitot et al. 1966 Scheuerbrandt 1965 Van Dyke and Szustkiewicz 1969 Leighton et al. 1968 Cadmus 1966; Chariot et al. 1966; Scheidt 1964; Strumeyer and Romano 1966; Wilding 1963 Scheuerbrandt 1965 Pitot et al. 1966 Stein et al. 1965 Kline et al. 1968 Lamy et al. 1967; Leighton et al. 1968; Pitot et al. 1966 Tappel 1968 Wilson et al. 1967 Gage and Litchfield 1966; Levine et al. 1965; Miwa et al. 1967; Winter 1960a Winter 1960b Pitot et al. 1966 Fleischer 1967; Siege1 and Cohen 1966; Willis et al. 1967 Schuel et al. 1964 Caniier and Gonnard 1966,1968 Roodyn 1965c Tappel 1968 Pitot et al. 1966 Pitot et al. 1966 Tappel 1964 Tappel and Beck 1967 Beck and Tappel 1967 Tappel and Beck 1967 Tappel and Beck 1967 Subiecl index P. 218

170 Enzyme assayed Glucokinase Glucose oxidase (glucose)

AUTOMATED ENZYME ASSAYS

References

Pitot and Pries 1964; Pitot et a1 1966, 1968 Cramp 1967; Getchell et al. 1964; Hill and Kessler 1961; Kawerau 1966; Logan and Haight 1965; Robin and Saifer 1965; Rosevaer et al. 1969; SaiferandRobin 1965;Tammesand Nordschow 1968 C lucose-6-phosphatase Leighton et al. 1968; Tappel 1964 Glucose-6-phosphate dehydrogHoober and Bernstein 1964; Miwaetal. 1967; Pitot et al. 1966, 1968 enase Glucuronidase Tappel 1964; Tappel and Beck 1967 Glutamate decarboxylase Camier and Gonnard 1966,1968; Leclerc 1967 Glutamate-oxaloacetate transAxelsson et al. 1965; Fingerhut et al. 1962; aminase (SGOT) Levine and Hill 1965; Moore and Sax 1969; Morgenstern et al. 1966b, 1967; Passen and Gennaro 1966; Schaffert et al. 1964; Schwartz et al. 1961; Trinder and Kirkland 1965; Van den Bossche 1965 Clutamate-pyruvate transaminase Axelsson et al. 1965; Levine and Hill 1965; (SGPT) SchaRert et al. 1964; Trinder and Kirkland 1965 Glutaniinase Pitot et al. 1966 Glutamine dehydrogenase Pitot et al. 1966 Glutamine synthetase Pitot et al. 1965 Glutathione reductase Kauppinen and Gref 1966 Glycerokinase Pitot et al. 1966 Guanase Nyssen and Dorche 1968 Hexokinase-glucose-6-phosphate Schersten and Tibbling 1968 dehydrogenase (glucose) Histidase Pitot and Pries 1964; Pitot et al. 1966 Histidine a-oxoglutarate Pitot et al. 1966 transaminase Hydroxybutyrate dehydrogenase Dube et al. 1968; Strandjord and Clayson 1966a L a-hydroxy acid oxidase Leighton et al. 1968 Lactate dehydrogenase (NADBerry and Walli 1966; Brooks and Olken 1965; linked) Capps et al. 1966; Dube et al. 1968; Hicks and Updike 1965; Hochella and Weinhouse 1965a, b; Hoober and Bernstein 1964; Houghton 1964; Levy et al. 1965; Morgenstern et al. 1965a, b, 1966a; Opher et al. 1966; Passen

PUBLICATIONS

Enzyme assayed

171

References

and Gennaro 1966; Pitot et al. 1966; Posen et al. 1967; Schwartz et al. 1961 ; Strandjord and Clayson I966a Lactate dehydrogenase (lactate) Hochella and Weinhouse 196% Lactate dehydrogenase (pyruvate) Cramp 1968; Minaire et al. 1966 D( -)-Lactate dehydrogenase Roodyn 1967a, b, 1969; Roodyn and Maroudas 1968 ~ ( )-Lactate -t ferricyanide Roodyn 1967a, b, 1969; Roodyn and Maroureductase das 1968 Leucine aminopeptidase Ratliff et al. 1966 Lysine decarboxylase (lysine) Schaiberger and Ferrari 1960 Lysozynie Burrows 1966; Jolles et al. 1967 Malate dehydrogenase Pitot et al. 1966; Roodyn 1965b Malic enzyme Pitot et al. 1966 Hill and Cowart 1966 Mutarotase B-N-acetyl-glucosaminidase Tappel and Beck 1967 Roodyn 1967a, b, 1969; Roodyn and MarouNADH dehydrogenase das 1968 Roodyn 1964, 1965b, c NAD-linked dehydrogenases NADPH dehydrogenase Roodyn 1969 Hill and Sarnmons 1965,1966 5’-Nucleotidase Ornithine carbanioyl transferase Girard et al. 1963; Strandjord and Clayson 1966b Pitot et al. 1965, 1966 Ornithine transaminase Oxidases Roodyn 1964 Papain Tappel 1938 Pepsin Vatier et al. 1966 Peptidases Lenard et al. 1965 Phosphatases Roodyn 1965c Phosphodiesterase Chersi et al. 1967 Phosphogl ucomu tase Hopkinson and Lewis 1967; Hoober and Bernstein 1964 6-Phosphogluconate dehydrogenase Cameron and Weg 1964; Pitot et al. 1968 Phosphohexoisomerase Schwartz et al. 1960b Polynucleotide phosphorylase Burns and Lazer 1965 Proteolytic enzymes Hazen et al. 1965; Heinicke et al. 1967 Pyruvate kinase Bigley et al. 1968 Rennin Nash et al. 1967; Skeggs et al. 1968 Barrera et al. 1969; Mundry 1965; Pitot et al. Ribonuclease 1966 SubiccI index p, 618

172

AUTOMATED ENZYME ASSAYS

Enzyme assayed

References

Serine dehydrase Succinate dehydrogenase

Pitot et al. 1966 Roodyn 1967a, b, 1969; Roodyn and Maroudas 1968 Tappel 1964; Tappel and Beck 1967 Clarke and Nicklas 1966 Stevens et al. 1967 Girard et al. 1966 a, b, c Pitot et al. 1966, 1968

Sulphatase Transglutaminase Transketolase Trypsin Tyrosine a-0x0-glutarate transaminase Tyrosine decarboxylase Tryptophan pyrrolase Tyrosine decarboxylase Urease (urea) Uricase (uric acid) Urocanase

Carnier and Gonnard 1968; Scheuerbrandt 1965 Pitot et al. 1966 Camier and Gonnard 1966 Wilson 1966 Barron and Bouley 1965 Pitot et al. 1966

APPENDIX I1

Terminology used in enzyme automation

ACCEPTOR STREAM (IN DIALYZER) - Stream, initially free of solute, that accepts solute across the dialysis membrane from the donor stream. AUTOMATED ASSAY - Enzyme assay in which the preparation of reaction mixture, addition of enzyme, incubation, and monitoring of reaction are performed automatically. AUTOSENSOR - Sensor attached to a recording device. CONCENTRATION OF ENZYME - Units/ml. CONTINUOUS FLOW ASSAY - Enzyme assay in which the fluid stream is moving continuously. DE-BUBBLER - Device for removing bubbles from gas-segmented stream. DILUTION FACTOR - Volume of reaction mixture divided by volume of reagent. DONOR STREAM (IN DIALYZER) - Stream containing solute that is to pass through the dialysis membrane. DISCRETE-SAMPLE ANALYZER - Method of automation in which the reaction takes place in separate vessels, often the sample cup itself. ENZYME MONITORING - Measurement of activity in a continuous stream of enzyme. ENZYME SAMPLE DETERMINATION - The automated determination of the activity in a number of enzyme samples. 173

Subjecl index P. 218

I74

AUTOMATED ENZYME ASSAYS

FALL CURVE (IN SAMPLING) - Part of the sample peak in which the sample concentration falls from steady state to zero. FLOW SYSTEM - Arrangement of all components of an autoanalyzer including the manifold. FRACTIONAL LINE VOLUME(FLV) - Flow rate of reagent line divided by total flow rate of all fluid lines. Reciprocal of linedilution factor. FRACTIONAL VOLUME - Volume of reagent divided by volume of reaction mixture. GAS-SEGMENTATION - Division of fluid by stream of bubbles, usually air. HALF-WASH TIME (W-5)- Time taken to change concentration at a given time to a concentration half-way between it and the final steadystate concentration. INTERRUPTED-FLOW ASSAY - Enzyme assay in which the reaction mixture is introduced into and removed from the reaction vessel by a flow procedure, but in which the reaction is monitored on a stationary reaction mixture. GRADIENT DILUTION FACTOR (GDF) - Concentration in effluent from gradient-making system/concentration of stock solution. LINE - Pump tube or pipette for introducing or removing fluid from an analyzer. LINE DILUTION FACTOR(LDF) - Total flow rate of all fluid lines/ flow rate of reagent line. LINE REAGENT - Reagent connected to pumping line in analyzer. MANIFOLD - System of pump lines, joints, mixing coils in Technicon autoanalyzer. MANUAL ASSAY - Enzyme assay in which none of the stages of the assay are automated. MOLAR CONVERSION FACTOR(MCF) - Concentration of solute in pmoles/ml that corresponds to an instrument reading of 1.0. MULTIPLE-ENZYME ANALYSIS (M.E.A.) - Automated system in which several enzyme-catalyzed reactions are measured. REACTION MIXTURE - Mixture of buffer, co-factors and substrate in which the enzyme-catalyzed reaction takes place.

TERMINOLOGY USED

175

REACTION RATE - Enzyme activity expressed in pmoles/min/ml reaction mixture. RISE CURVE (1N SAMPLING) - Part of sample peak in which the sample concentration approaches steady state. SAMPLE CARRY OVER - Per cent carry over of one sample to the next (100 - WE). SAMPLE PATTERN - Arrangement of solutions in the sampler (important in multiple-enzyme analysis with recycling groups of substrates). SEMI-AUTOMATED ASSAY - Enzyme assay in which some, but not all, of the assay procedure is automated. SENSOR - Device for following the progress of an enzyme-catalyzed reaction. SINGLE-ENZYME ANALYSIS (S.E.A.) - Automated system in which only one enzyme is measured. SPECIFIC ACTIVITY - Enzyme activity expressed in pmoles/min/mg protein. STOCK REAGENT - Reagent used to prepare line reagent by suitable dilution. TOTAL REACTION - Enzyme activity expressed in pmoles/ml reaction mixture in a given time (not necessarily 1 min). TOTALLY AUTOMATED ASSAY - Enzyme assay in which the preparation of the reaction mixture, addition of enzyme, incubation monitoring of the reaction, calculation and presentation of results are automated. UNIT (U) - Amount of enzyme that catalyzes the transformation of 1.0 pmole of substrate per minute. WASHING EFFICIENCY (WE) - Per cent return from steady state to zero concentration in time interval between samples.

Subiecl inifex P. 218

APPENDIX 111

Apparatus used in enzyme automation

III.I . Analytical systems and instruments The reader is referred to Analytical Chemistry (1968) Vol. 40,No. 9, for a guide to laboratory equipment that may be relevant to enzyme assays. The following account of some equipment of use in enzyme automation was completed in October 1969 and is not intended to be comprehensive. New products are appearing on the market at an increasing rate, and established instruments are undergoing constant modification and improvement. In such a situation, it is probably best to present a general survey indicating the type of equipment that has been developed. It is convenient to discuss representative examples of each type of system in more detail. However, it should be stressed that this does not imply that the systems described are necessarily the best. Similarly, the omission of any equipment should not be taken as an implied criticism of its value. (It is, in fact, difficult to obtain up-to-date information on all the equipment on the market.) The description of equipment and the terms used for the various components are based on brochures supplied at the time by the manufacturers, and the author trusts that the task of condensing this literature has not been done in a misleading or unfair manner.* To conclude, the following account is merely intended as an introductory guide to the reader, and is not a critical asessment of current equipment.

* Every attempt has been made to present the characteristics of the instrument accurately. However, improved models are constantly appearing, and the reader is strongly recommended to consult the manufacturers for more direct information. 176

APPARATUS USED

I77

III.1 . 1 . Automated andytical systems A variety of systems is now available for automated analytical chemistry in general. Many have been designed primarily for clinical chemistry, but some are of a more general nature. A few have been developed specifically for enzyme assays, and these will be described first. Bausch & Lomb market the ‘ZYMAT 340’. This is a discrete-sample analyzer designed specifically for enzyme studies, particularly enzymes of clinical importance with assays based on following variations in the concentration of reduced pyridine nucleotides at 340 nm. The instrument contains two concentric wheels which move synchronously. The smaller Sample Wheel fits directly over a larger Cuvette Wheel, with a capacity of 47 cups. Enzyme solutions such as serum samples, are loaded into the Sample Wheel, and appropriate volumes are transferred into reaction vessels in the Cuvette Wheel by a sample probe. Reagent is added and after temperature equilibration, the appropriate substrate is added from another probe and the reaction mixture is rapidly mixed by an air jet. The reaction vessel itself acts as a cuvette and moves into the light beam of a filter colorimeter after an appropriate time delay. The result is finally directly printed out in digital form in international enzyme units. The instrument may also be monitored by an appropriate strip chart recorder. The 47 samples can be analyzed in 108 min. Various other automated enzyme analyzers have been described in the literature, for example the ‘ASTRA Enzyme Assayer’ (see Schwartz and Bodansky 1963). As a part of the general ‘MECOLAB’ system (see below) Joyce Loebl market an instrument called the ‘ENZYMAT’ which is specifically designed for the determination of enzyme rates. 15 reaction mixtures may be monitored each minute in a rotating tube carrier. The reaction mixtures are contained in special mixing tubes which also act as cuvettes of the colorimeter. Automatic micropipettes and syringes dispense the enzyme and reagents into the mixing tubes at appropriate times, and the results are recorded on a special drum recorder which operates in synchrony with the rotating tube carrier. Cotlove et al. (1967) have described an ‘ENZYME ,ANALYSIS SYSTEM (E.A.S.)’ designed to assay certain clinically important Subjecr index p . 218

178

AUTOMATED ENZYME ASSAYS

enzymes. The analyzer is a discrete-sampling system. It measures absorbance at 340 nm for 12 programmed intervals after the addition of substrate. An analogue/digital converter passes the signal to an online computer which calculates the rate of increase in absorbance by the method of least squares. The results are converted into appropriate enzyme units and the computer also compiles laboratory and patient reports. The sampling rate is 80/hr and the sample tubes are placed in machine-readable carriers. The probe is thoroughly rinsed between samples in order to reduce cross-contamination. The system can operate in two ways : single mode for determination of lactate dehydrogenase, and dual mode to determine glutamate/oxaloacetate aminotransferase and glutamate/pyruvate aminotransferase. Another device for the measurement of enzyme rates is the LKB 'REACTION RATE ANALYSER. It is a discrete-sampling system that operates with movable racks containing up to 10 sample tubes. These act both as reaction vessels and cuvettes of the colorimeter. Enzyme and reagent are added at appropriate points to the racks, and after the reaction mixture is prepared, the rack is stopped and the progress curve measured for 1-9 min. The colorimeter system uses filters and can operate at 340 nm. Most of the systems to be described in this section are based on the discrete-sampling principle. The most notable exception is the Technicon system, which has been discussed fully in the main text of this book. In summary, it is a modular system for continuous flow wet chemical analysis. The arrangement of each assay system (or manifold) is based on connections of pump tubing, transmission tubing, various coils and junction pieces and any of the following main modules that may be required : sampler, proportioning pump, dialyzer, heating bath, colorimeter or spectrophotometer, recorder, fluorimeter, flame photometer, continuous filter, data logger, scale expander and programmer. Samples from the sampler module are pumped through glass or plastic tubing and smearing of the sample peak is greatly reduced by using gas-segmentation, i.e., a train of bubbles, usually air. The usual analytical system for enzymology consists of a sampler, proportioning pump, heating bath, dialyzer or continuous filter (if protein interferes

APPARATUS USED

179

in the assay) and a colorimeter, spectrophotometer or fluorimeter. The assays may be run with enzymes in the sampler (single-enzyme analysis, S.E.A.) or a range of substrates in the sampler (multipleenzyme analysis, M.E.A.). In several published systems, the Technicon apparatus has been connected to a Gilford 2000 multiple-sample absorbance recorder (see below). The rate of analysis is between 20-70 samples/hr, depending on the system. In addition to thegeneral systems described above, Technicon have multi-channel analyzers specifically designed for clinical analysis, using the principle of sequential multiple analysis (S.M.A.). Some of the channels in these systems are for enzyme tests. Other firms have produced relatively large scale complex multichannel analyzers for clinical work. For example the AGA Medical Division have developed the ‘AUTOCHEMIST’ (see Jungner and Jungner 1968). Assays available include glutamate/oxaloacetate aminotransferase, glutamate/pyruvate aminotransferase, acid and alkaline phosphatase, amylase, lipase and lactate dehydrogenase. A conveyor system carries samples to a Central Chemical Processor which is a multi-channel discrete-sampling system. Small units, called ‘satellite stations’ are used for occasional assays on those not suited to automation, and these stations are connected to the main system. The operations are controlled by an Electronic Control Unit which includes a PDP/8 digital computer working on time with the Central Processor. There are 24 analytical channels, each capable of about 130 samples/hr, so that the total rate of analysis can be as high as 3000 samples/hr. A sample identification system prevents errors in sampling and the computer performs full data processing of individual results and clinical records. The Vickers ‘MULTICHANNEL 300’ is another large scale multichannel analyzer designed for clinical biochemistry. Available enzyme assays include alkaline phosphatase, glutamate-oxaloacetate aminotransferase and lactate dehydrogenase. The machine is of modular construction and the reactions are carried out by discrete-sampling turn-tables, automatic pipettes and probes in ‘reaction rotor consoles’. Up to 12 channels can operate simultaneously. The samples enter via a Subiecr index P. 218

I80

AUTOMATED ENZYME ASSAYS

magazine-feed system, and a ‘primary transfer diluter’ then transfers appropriate volumes to the distributer assembly and then to the appropriate reaction console. After the reaction has taken place, the reaction mixture is transferred to a double-beam colorimeter, with appropriate blank adjustment, or to a flame photometer. The system includes facilities for sample identification and data processing. The reaction vessels are washed out after use by a ‘tube laundry’ and the machine can operate at 300 samples/hr. Another modular analytical system is ‘MECOLAB’ from Joyce Loebl. The modules include discrete-sampling reaction vessels of turn-table design, an automatic centrifuge, colorimeter, flame photometer, a tape-controlled laboratory programmer which can control up to 16 laboratory components (such as pumps, heaters, valves and mixers) and a data-processing system with digital output. One module, the ‘ENZYMAT’, has been described above. There is now a considerable number of discrete-sampling systems on the market of varying complexity and space does not allow a complete description of each case. The Cecil ‘CE404 COLORIMETER SYSTEM’ is a modular arrangement for automatic colorimetry. A variety of assemblies are possible, and the more complex system contains a sample changer with programmer, pumps, reservoirs and a system for transferring the sample to the automatic filter colorimeter with its own direct meter as well as a recorder. 30 samples on the sampler turn-table can be measured in 10 min. Samples can also be read directly in the colorimeter in matched tubes, so that the system can be run as a ‘mixed’ manual and automatic analyzer. Grant have marketed the ‘GRANT LINSON AUTOLAB’ which is a discrete-sample analyzer with a linear train of tubes connected by plastic holders to form a ‘specimen chain’. The system includes automatic syringes and probes and measurement is with a photometer, data converter and digital print-out. Griffin & George have produced the ‘GRIFFIN EEL BIOANALYST’ which is another discrete-sample analyzer using a colorimeter with digital print-out facilities. It can operate at 120 samples/hr and has facilities for automatic standardization and blank compensation. The ‘CLINOMAK’ system from Camlab is a

APPARATUS USED

181

discrete-sample analyzer based on the turn-table and probe principle, again with an automatic colorimeter. U p to 90 samples can be assayed at a time and the sample volume can be between 20 and 200 pl. There are probes for adding up to five reagents automatically. A similar system is the ‘AC AUTOMATIC ANALYTICAL SYSTEM’ from Pye-Unicam. In this, the sample is returned to the reaction vessel after it has been read. The timing sequence and reagent volumes are controlled by preformed key-plates that are inserted into the instrument. The system can operate at 120 samples/hr. One of the earlier discrete-sample analyzers is the ‘ROBOT CHEMIST’ from Warner-Chilcott (see Morgenstern et al. 1967). A ‘master controller’ coordinates the operation of the instrument and up to seven reagents may be added by automatic pipettes. The reaction takes place in test tubes mounted in a circular sample train. Probes empty the reaction mixture into a spectrophotometer and the reading is converted to digital form with a data converter. Tubes are washed and rinsed automatically. In the ‘BTL ANALMATIC’ system from Baird & Tatlock the 100 reaction tubes are held in a batch frame in a water bath. Reagents are dispensed from automatic pipettes which traverse the rack from above. After the reaction, the fluid is pumped into a double-beam colorimeter or twin-channel flame photometer. The system includes a linearized and digital print-out unit. The enzyme assays reported include alkaline phosphatase and glutamateoxaloacetate aminotransferase. Up to 300 samples/hr can be analyzed with a minimum sampling volume of 10 PI. The ‘617 AUTOMATIC ANALYZER’ from Quickfit & Quartz is another discrete-sample system, which performs the following operations automatically. Sample is taken from one turn-table and mixed with appropriate diluents and reagents. If a precipitate is formed (as in the de-proteinization of sera), this can be removed by an automatic centrifuge (‘microcentrifuge’) which consists of a rotating disc with probes that move to the centre or periphery as the sample is centrifuged. The clear supernatant is passed to a second turn-table, and the sediment is removed from the centrifuge by a vacuum pump. The centrifuge is then rinsed for the next sample. A sample is taken from the superSubject index p . 218

182

AUTOMATED ENZYME ASSAYS

natant, mixed with appropriate diluents and reagents and after the reaction is complete the fluid is transferred to an automatic colorimeter with digital print-out. The analysis rate is approximately 100 samples/hr and micropipettes up to 500 pl in volume pick up the sample. Diluter and dispenser syringes can deliver up to 5 ml per reagent.

III. 1.2. Spectrophotometers, colorimeters und their accessories Enzymology has relied heavily on measurement of changes in light absorption and most spectrophotometers or colorimeters are of direct use in enzyme assay. Almost all instruments can be adapted to give automatic recording of the changes in absorbance and there is a very wide range of instruments available from simple direct-reading colorimeters to complex high-precision spectrophotometers. Some instruments have been specifically designed for enzyme assay and a recent tendency to add sampler modules, automatic cell changers and program controllers has resulted in instruments that are virtually automatic analyzers of the type described above. In fact the use of modular systems has made it increasingly difficult to classify equipment used in enzyme automation since there is, in effect, a continuous spectrum from totally automated to manual systems. However, spectrophotometers and colorimeters constitute a fairly clear class. The Beckman ‘KINTRAC VII’ is designed specifically to monitor kinetics of chemical reactions, with particular emphasis on enzyme analysis. The automatic cell compartment can take 7 cuvettes. These have ‘in-situ ports’ for addition of reagents. Since the cuvettes act as the reaction vessels, it is convenient that they can be stirred magnetically during the course of the reaction, particularly in experiments involving cell particles of turbid solutions. The reaction is followed by the spectrophotometer and output is onto a 10 inch recorder with linear read-out. However, there is also a direct read-out meter calibrated in yo T and absorbance units (0-2). Controls in the instrument include facilities for photometric scan and zero suppression, selection of appropriate samples from the seven cells, alteration of the time the sample cell is in the light path of spectrophotometer and of the time between readings. Temperature control is from an external thermo-

APPARATUS USED

183

circulator, and there is also a digital concentration converter giving direct read-out of concentration, and correcting electronically for deviations from the Lambert and Beer law. The Gilford ‘MODEL2000 MULTIPLE SAMPLE ABSORBANCE RECORDER’ is an instrument that is used in conjunction with suitable monochromators to give a linear direct reading spectrophotometer. It is designed for high stability for measurements at fixed wavelengths and it can be adjusted to give full-scale deflection for any absorbance value between 0.1 and 3.0. I n addition, the rccorder may be adjusted to zero at any absorbance value up to 3.0. An automatic cuvette positioner takes up to four cuvettes or flow cells, and it can move from one position to the next in approximately 1%sec. The dwell time in the light beam can be varied from I to 45 sec. An automatic blank compensator is available to correct for base-line drift, and there is a dual-wavelength mechanism available for measuring at alternate wavelengths. Recently, Pye-Unicam have produced the ‘SP 3000’ automatic UV spectrophotometer that also has many features of use in enzyme assay. A cell correction control is used to eliminate differences between cells, and there is an arrangement for automatic calibration between each cycle of readings. During the assay the readings are presented on a four-digit illuminated display in either 0-1 12.4% T or -0.050 to 2.000 absorbance units. A digital-printer accessory types out each result with an appropriate reference number. An automatic sample changer holding 50 samples is included in the instrument, and for assays involving a small number of samples, a sampling head allows the introduction of solutions into the cells without removing them from the instrument. Each sample can be measured at up to 5 different wavelengths with the normal automatic wavelength selector, or up to 10 wavelengths with an additional unit. A programmer timer controls the cycle of operation of the instrument. 28 or 30 sec are normally required per reading, but a response time of 12 sec is possible with a suitable accessory. The SP 8000, another recent instrument, also provides digital print-out and results in units of concentration. Zeiss market a modular system for photometric analysis, and the basic spectrophotometers or filter photometers can be operated with Subjecr index P. 218

184

AUTOMATED ENZYME ASSAYS

several different modules. These include sample changers, a variety of thermostatically controlled cell holders, automatic sample units, a turbidimetric fitting, a stirring device, an automatic cell unit with controller, a transmittance-absorbance converter and recorder. Photometric measurements use either a photocell or photomultiplier, and the sampling units may either be operated manually or under program control. The automatic cell unit can hold up to six cells with a path length of up to 5 cm. The AMINCO-CHANCE DUAL WAVELENGTH SPECTROPHOTOMETER marketed by American Instrument Company is an instrument specifically designed to make measurements of small changes in absorbance in turbid suspensions, for example in studies of sub-cellular particles, particularly mitochondria. Two beams of light of different wavelength pass alternately through the sample, with one beam acting as reference for the other. This eliminates small fluctuations in absorption of light due to changes in the turbidity of the sample during the assay. Accessories to the instrument include a rapidmixing chamber for following fast reactions by the stopped-flow method, a side-illumination attachment for studies on photosynthesis, a fluorescence attachment and a scanning attachment to scan the wavelength of one of the beams against a fixed reference wavelength. The last accessory is of use in the measurement of absorption spectra, for example of the cytochrome system, in turbid suspensions. The STOPPED-FLOW SPECTROPHOTOMETER marketed by Durrum is used to study rapid kinetics of chemical reactions. The two reaction components are placed in syringes, and a flow activator forces each component rapidly through a mixing jet into the observation chamber, which is the cuvette of the spectrophotometer. The plunger is stopped, bringing the flow to a halt, and the reaction is measured in an evironment free of turbulence. It is followed on an oscilloscope with a recorder camera. It is possible to vary the wavelength, the band width, the temperature, the time of incubation, the volume of the sample and the sensitivity of the instrument. Within 2 msec, mixing is 99% complete. The volume of the mixingjet to the centre of the flow cuvette is 60 pl, and 0.2 ml or more of each reaction component may be used.

APPARATUS USED

185

It is impossible to give a comprehensive account of the range of general purpose spectrophotometers and colorimeters on the market. A few characteristic instruments will be briefly described and the names of several firms with their products briefly listed. The ‘SP 500’ spectrophotometer by Unicam is a single-beam instrument with a programme-controller unit that enables four standard cells or semimicro cells to be measured at a fixed wavelength at predetermined time intervals, with a constant temperature cell holder. Various accessories include a linear/logarithmic recorder and a fluorimeter attachment. Perkin Elmer market a variety of instruments of use in enzyme analysis. The ‘MODEL 402’ spectrophotometer is a scanning UV instrument with a four-cell unit controlled by a programming unit. The ‘MODEL 124’ has a digital-concentration read-out accessory, with a correction system for assays that do not follow the Lambert and Beer law. An averaging system improves the precision of assay by averaging 4, 8 or 16 readings of the same sample. We may now merely list some firms, with some of their relevant equipment, apart from that mentioned above. Bausch & Lomb market spectrophotometers, pen recorders and a concentration computer attachable to a spectrophotometer. Beckman market a range of spectrophotometers, a sample changer and a concentration converter. Canal Industrial Corporation market spectrophotometers, and Carey provide a range of spectrophotometers, an automatic cell changer, data-logging equipment and other accessories. Durrum market a prism-grating spectrophotometer, and Elvi market spectrophotometers and an automatic recording microspectrophotometer. Gilford have a range of spectrophotometers, automatic cuvette positioners, a high-speed sampler, a digital absorbance/concentration meter and a data lister. A scanning dual-wavelength spectrophotometer is available from Phoenix Precision and recording and digital spectrophotometers are available from Shimadzu Seisakusho. In addition to spectrophotometers, there is a very wide selection of filter colorimeters that are, of course, considerably cheaper. The Zeiss filter photometer has been mentioned above. Eppendorf market a filter colorimeter that gives readings proportional to absorbance, with Subiecr index P. 218

186

AUTOMATED ENZYME ASSAYS

a compensation system for reading dense or turbid solutions. Accessories include an automatic cuvette changer with recorder, integrator and calculator. Up to 6 cuvettes are carried, and the cell holder is readily replaced with new cuvettes. Fisons have marketed the ‘VITRATON DIGITAL COLORIMETER. A semi-automatic system transfers the sample from a test tube rack to a colorimeter with a small volume flow cell (80 PI). The colorimeter is connected to an analogue/digital logarithmic converter and thence to a printer. The output is in absorbance units (0-0.099) but ‘multiplication modules’ may be plugged in to convert the absorbance into appropriate units of concentration. Carry-over is reported to be less than 1% for a 2.5 ml sample and each sample can be transferred and measured in 12 sec. Technicon market single or double continuous-flow colorimeters using filters, and direct-reading colorimeters are available from many firms, including Watson Marlow, for example. Evans Electroselenium market an automatic colorimeter and sampler (EEL models 171 and 178). The ‘ESKALAB’ Clinical Chemistry system (Smith Kline Instrument Co.) is an intermediate between fully automated systems and manual methods and is a good example of a ‘work-simplified’ technique. The system operates as follows: special reagent tablets are supplied for the various assays available, which include alkaline phosphatase and serum glutamate-oxaloacetate aminotransferase. The tablets are dropped into disposable plastic cuvettes and the required volume of diluent is added from plastic reservoirs which are attached to the cuvettes with adapters. The colorimeter has a built-in, dry-well incubator into which the cuvettes are placed for temperature equilibration and for the actual assay. Enzyme (serum) samples are added to the cuvette assembly from disposable micropipettes and the reaction is followed by transferring the cuvette from the incubator to the cell holder of the colorimeter. The latter is a double-beam system designed for high stability and giving a linear response over the range 0-1.5 absorbance. Sampling, diluting, filtering or centrifuging of the reaction mixture necessarily limits the rate of analysis. If one simply wishes to measure the absorbance in a previously prepared sample, one can clearly

APPARATUS USED

187

operate at much faster rates. LKB have produced a ‘HIGH SPEED CALCULATING ABSORPTIOMETER’ for this purpose. The device can measure the absorbance of 1500 samples/hr. They are loaded in movable racks in disposable polystyrene or glass cuvettes. Blank and reference values are entered into the instrument and the samples rapidly passed through the light beam of the absorptiometer. The output is displayed visually on 6 display tubes, and is also printed digitally with sample identification, and an appropriate code number for the analysis performed. The high speed of analysis is achieved by using the sample tube as the cuvette of the sensing system, so that no fluid transfer is needed.

III. 1.3. Other automatic sensing devices There are, of course, many ways of following enzyme-catalyzed reactions other than by spectrophotometry. Fluorimetry is an important technique, and a variety of instruments are available (e.g. from American Instrument Co., Baird Atomic, Beckman, Farrand, Perkin Elmer, and Technicon). The first of these companies sells the ‘AMINCOBOWMAN’ spectrophotofluorimeter. It is an instrument that can both activate and measure fluorescence in the ultraviolet and visible regions of the spectrum. Light from a xenon lamp is dispersed by a grating monochromator and is used to excite the sample. The fluorescent light so produced passes through a second grating monochromator to the detection system, and the output is displayed on a cathode ray oscilloscope or pen recorder. Accessories include an automatic cell-changer specifically designed for enzyme assays, with 4 cells in a rotating turret, and special recorders for following rapid kinetic changes. A variety of electrodes and appropriate recording systems are used to follow enzyme reactions and again it is not possible to describe the full range of equipment available. Electrodes are available from many firms (for example E.I.L. and Beckman) and are used to measure p H , a variety of ions, COz and Oz. Oxygen electrodes are sold by many firms (e.g. Rank) and some firms market systems for polarographic recording. The ‘OXYGRAPH’ from Gilson records rapid changes in oxygen concentration by this method. Two systems are used. For Subject rndex a. 118

188

AUTOMATED EYZYME ASSAYS

solutions with conductive buffers, a rapidly oscillating platinum cathode is used. If gases or solutions with non-conducting buffers are studied, a micro-Clark electrode is used. The microplatinum cathode oscillates a t 6000 cycles/min in the test solution, and is coupled by a salt bridge to a calomel reference anode. The Clark electrode has a platinum cathode and a silver reference anode. It is covered with a thin Teflon film to allow diffusion of oxygen. A polarizing voltage of 0.6 to 0.8 V is applied to both systems. The current limited by the oxygen concentration is measured and appropriately recorded. The instrument also has a system for simultaneously measuring oxygen concentration and pH. The ‘BIOLOGICAL OXYGEN MONITOR’ from Yellow Springs also used the Clark electrode inserted into a temperature controlled assembly. There are two electrodes, so that one can be used while the reaction mixture for the second is being prepared. Sargent Welch also market a recording polarograph. Many improved manometric systems are now available. Manometric apparatus and accessories are available from a variety of firms (e.g. Braun, Bronwill, Gilson and Shandon). The Gilson ‘DIFFERENTIAL RESPIROMETER is a constant-volume respirometer with digital readings for each flask. The units that give the readings are called ‘volumometers’ and are essentially a combination of a manometer and a micrometer. The changes in volume of gas in the flasks are given directly in pl. Gilson also market a recording respirometer. The ‘LONGATOR recorder, from Braun, is a device for recording pressure changes in up to 6 constant-volume respirometers. It is designed to operate with any type of Warburg apparatus and can measure pressures of up to 300 mm of water, in 6 cycles of 50 mm. Space does not allow a complete description of all automatic sensing devices of use in enzyme assay. A recent instrument to measure heat changes should be mentioned howevei. It is the LKB ‘MICROCALORIMETRY SYSTEM’ which measures the heat changes that can occur during enzyme-catalyzed reactions. A flow and a batch microcalorimeter are available. A twin-calorimetry system is used to eliminate non-specific heat changes, i.e., the changes in the reaction vessel are balanced against those in a ‘blank’ vessel containing non-reactant

APPARATUS USED

189

liquid. The heat liberated during the reaction flows to a heat sink through thermopiles. The response from these is appropriately balanced, amplified and recorded.

III. 1.4. Dispensers, diluters, pumps and sampling s y s t e m An increasing number of devices are coming onto the market to simplify and automate the processes involved in diluting, mixing and delivering measured volumes of samples or reagents. These may be used to elaborate so-called ‘work-simplified’ systems. Again, space does not allow a full description of the equipment currently available for use in enzyme assays. Some of the equipment and firms, however, may be given as follows. Various automatic diluters and dispensers are available, for example the ‘AUTOSPENSER’ and ‘AUTODILUTOR of American Optical, the ‘DILUTRONIC 250’ of Baird & Tatlock, a variety of autodiluters, dispensers and automatic probes from Fisons, an automatic diluter called ‘DILUSPENCE’ from Griffin & George, and automatic pipettes from Eppendorf, from Flaig and from Schwartz. Hook and Tucker supply a variety of devices for diluting and dispensing one or several reagents, using syringes with appropriate valves to prevent back-flow. Their ‘SAMPLE PREPARATION UNIT’ consists of two concentric rings of sample bottles on a turn-table, with a mechanism for transferring samples from one ring to the other, and for diluting the samples. The samples can be transferred from the sample-preparation unit to a colorimeter or fluorimeter for measurement. The Gilson ‘TRANSFERATOR’ can transfer samples from test tubes into a spectrophotometer cuvette. It then empties and refills the cuvette, and can be operated by a foot switch. Thomas market automatic sampling and print-out systems for spectrophotometry. As mentioned above, many colorimeters and spectrophotometers have sampling units and there is a wide choice of equipment in this field. There is also a wide selection of pumps of use in automated enzyme analysis. The following may be mentioned as representative of the range of instruments available. Biotec and LKB provide peristaltic pumps for fermentation and chromatography systems. A ‘DIAL-APUMP’ multi-channel pump is available from Durrum, and Horwell subjecr index P. 218

190

AUTOMATED ENZYME ASSAYS

supply the ‘BUCHLER POLYSTALTIC’ Cchannel pump. Multichannel pumps are also available from Hughes, Technicon and from Watson-Marlow. Some pumps are of particular use in making gradients, and Phoenix Precision market a variable-gradient pump, the ‘VARIPUMP’. The above survey is by no means comprehensive and is an attempt to provide an overall picture of the kind of equipment currently marketed for automated enzyme assays. In particular no attempt has been made to describe ancillary data-processing equipment such as analogue-to-digital converters, paper-tape punches, magnetic-tape recorders or on-line electronic calculators used to convert instrument readings to units of concentration. An ingenious multiple-cuvette rotor has recently been described by Anderson (1969) and has great potential as a new approach to biochemical automation. No detailed procedures for performing specific enzyme assays have yet been published but these are awaited with interest. The analytical system based on the rotor (‘General Medical Sciences-Atomic Energy Commission’ or ‘GEMSAEC‘) is available in prototype form from a number of firms including Electro-Nucleonics. 111.1.5. Reagents

In parallel with the obvious development of automated enzyme assay systems, several chemical firms provide convenient preparations of reagents for specific enzyme assays. BDH have recently announced clinical assay sets for several enzymes, Boehringer sell test-kits for enzyme assays, Worthington have a range of special enzyme reagents and the American Monitor Corporation sells standard reagent kits specifically for automated enzyme assays. The ‘ESKALAB’ reagent tablets have been mentioned above (p. 186). There is little doubt that the next 5-10 years will see a great expansion in the range and sophistication of assay systems on the market, and it is hoped that manufacturers of analytical instruments will work in closer liaison with the firms producing biochemical reagents so that automated enzyme assays will be performed with greater ease.

APPARATUS USED

191

III. 2. Addresses offirms (NB : Some firms have addresses in several countries and in these cases the reader should refer to the address below for full details of the addresses in various countries; in general the parent company’s address has been given, but sometimes the U.K. subsidiary has been given.) AGA Medical Division, Lidingo, Sweden American Instrument Co., Inc., Georgia Ave., Silver Springs, Maryland, U S A . American Monitor Corporation, P/O Box 40396, Indianapolis, Indiana, U S A . American Optical Corporation, South Garrard Boulevard, Richmond, California, U.S.A. Baird-Atomic Ltd., Station Lane, Hornchurch, Essex, England Baird and Tatlock (London) Ltd., Freshwater Rd., Chadwell Heath, Essex, England Bausch and Lomb, Bausch St., Rochester, New York, U S A . BDH Chemicals Ltd., Poole, Dorset, England Beckrnan Instruments, International SA, Rue des Pierres-du-Niton, Geneva, Switzerland Biotec AB, P/O Box 16152 Stockholm 16, Sweden Boehringer, GmbH, Mannheim, W. Germany Braun, B., Mekungen Apparatebau, W. Germany Bronwill Scientific, Rochester, New York, U.S.A. Camlab (Glass) Ltd., Milton Rd., Cambridge, England Canal Industrial Corporation, Fisher Lane, Rockville, Maryland, U S A . Carey Instruments, Monrovia, California, U S A . Cecil Instruments Ltd., Green End Rd., Cambridge, England Durrum Instrument Corporation, East Meadow Drive, Palo Alto, California, U.S.A. E.I.L., Richmond, Surrey. England Electro-Nucleonics Inc., Passaic Ave., Fairfield, New Jersey, U S A . ELVI, P. Za. G. Cecare, Milan, Italy Eppendorf Geratebau, Netheler and Hinz Gmbh., Hamburg, W. Germany Evans Electroselenium Ltd., Halstead, Essex, England Farrand Optical Co., Inc., Mount Vernon, New York, U S A . Fisons Scientific Apparatus Ltd., Lougborough, Leicestershire, England Flaig and Sons, Exelo Works, Ramsgate, Kent, England Gilford Instrument Laboratories, Inc., Oberlin, Ohio, U S A . Gilson Medical Electronics, Villiers-le-Bel, France Subjecr index p . 218

192

AUTOMATED ENZYME ASSAYS

Grant lnstruments (Cambridge) Ltd., Barrington, Cambridge, England Griffin and George Ltd., Wembley, Middlesex, England Heath Co., Benton Harbor, Michigan, U S A . Hook & Tucker Ltd., Brixton Rd., London, England Horwell Ltd., Kilburn High Rd., London, England Hughes and Co., Ltd., Epsom, Surrey, England Joyce, Loebl and Co., Ltd., Princesway, Team Valley, Gateshead, Co. Durham, England LKB Produkter AB, Bromma, Sweden Perkin Elmer Corporation, Norwalk, Connecticut, U S A . Phoenix Precision Instrument Co., Philadelphia, Pennsylvania, U S A . Pye-Unicam Ltd., York St., Cambridge, England Quickfit and Quartz Ltd., Staffordshire, England Rank Bros., Bottisham, Cambridge, England Sargent-Welch Scientific Co., Foster Avenue, Chicago, Illinois, U.S.A. Schwarz Bioresearch, Orangeburg, New York, U.S.A. Shandon Scientific Co., Ltd., Pound Lane, Willesden, London, England Shimadzu Seisakusho Ltd., Chiyoda-Ku, Tokyo, Japan Smith Kline Instrument Co., Welwyn Garden City, Hertfordshire, England Technicon Corporation, Ardsley, New York, U S A . Thomas, Arthur, H., Co., Vine St. at 3rd, Philadelphia, Pennsylvania, U S A . Vickers Ltd., Millbank, London Warner-Chilcott Instruments Division, Richmond, California, U S A . Watson-Marlow Ltd., Marlow, Buckinghamshire, England Worthington Biochemical Corporation, Freehold, New Jersey, U S A . Yellow Springs Instrument Co., Yellow Springs, Ohio, U S A . Zeiss, Oberkochen/Wuertt, W. Germany

APPENDIX IV

Computer program for generalized enzyme automation system

The Program is in FORTRAN and has been run on the University of London ATLAS Computer, using instructions in ATLAS FORTRAN V Manual (Schofield 1967).

I V .I. Input: general instructions Input is on IBM cards with 80 columns per card. The program is arranged in sections which must be entered in the correct sequence. Entry into each section is by the following key words (in their correct order of entry); INTRODUCtion, MOLAR COnversion, INCUBATIon, LINE VOLume, LINE REAgent, STOCK Solution, GRADIENT ASSAY Conditions, PROTEIN, SAMPLE Pattern and RESULTS. Only the first eight letters or spaces are required but it is convenient to punch the entire key word for checking data. With double words (e.g. LINE REAgent) there must be a single space between words. All key words are entered on the left-hand side of the card, starting at column 1. Unless overwritten, data stored in a given section will be used for calculations in any subsequent sections if required. Thus once a given set of molar conversion factors are calculated, they will be used for all subsequent calculations until over-written. Care should be taken in over-writing data. For example, if 20 values are entered on the Grst occasion n n d only 15 are over-written, the remaining 5 will be used for any subsequent calculations involving 20 values. One may proceed 193

Subject index P. 218

194

AUTOMATED ENZYME ASSAYS

from any section to any other section further down the above list, i.e., one may omit any section. The RECYCLE instruction returns control to the beginning of the program and can be used to overwrite data in previous sections. For example, if all the sections have been entered in the first assay and a different incubation time has been used in a second assay, one would use the following sequence: INTRODUC

I

RESULTS (assay No. 1) RECYCLE INCUBAT1 RESULTS (assay No. 2) FINISHED (The term FINISHED is placed at the end of the last assay.) For a series of assays carried out under identical conditions with a different enzyme sample in each assay, one would use the sequence: INTRODUC

I

PROTEIN (first enzyme sample) SAMPLE P RESULTS (with first enzyme sample) RECYCLE PROTEIN (second enzyme sample) RESULTS (with second enzyme sample) RECYCLE etc. The system thus allows for flexible use of the program, but also ensures that data cannot be entered in the wrong sequence. Textual comments may be entered at many points in the program and may be on any number of cards (including zero) with characters entered in any part of the card (‘unrestricted input’). Comments are terminated by a new card with ZZ in columns 1-2 (or alternatively with the instruction LASTWORD in columns 1-8). In some parts of the program the ZZ card is obligatory since it is assumed that textual material will always

195

COMPUTER PROGRAM

be inserted. Elsewhere, however, insertion of comments is optional and the instruction PRINT is used to determine whether the data will be printed or merely stored for further use. In these cases, if the PRINT instruction is omitted, there is no provision for comments and the ZZ card should be also omitted. In the detailed instructions below, unless otherwise specified, numbers are entered in up to 10 columns with up to four decimal places (the F 10.4 format specification in FORTRAN). Instrument readings and blanks for both the gradient calibration and the enzyme assays are entered in extinction values (log,, I,/Z) in 8 columns per value, with up to three decimal places (F 8.3). This gives 10 readings per card, which is a convenient number for checking. After the last of such values, any number greater than 5.0 (e.g. 9.9) is entered to terminate the readings. In this way any number of readings may be entered. Thus three hypothetical readings would be entered as: ssso. I3sssO.391 sssl.0l5sssss9.9 where a single space is represented by ‘s’. Integer values are marked (1) and should fill the field starting from the right, i.e., for (1 3) the values 9 would be entered as 009. In some cases, sets of values are entered by cycles of the same input instructions. These are marked by vertical arrows thus :

+I

Terms in capital letters should be entered in the columns specified. Entries in new cards all start in column 1.

I V.2. Input: detailed instructions INSTRUCTION

COLUMN NO.

LISTDATA Enter any material that requires printing here (any number of cards may be used with entries (i.e., unrestricted input’ anywhere on the card).

1-8

ENDLIST

1-7

1-80

Subject index P. 218

196

AUTOMATED ENZYME ASSAYS

INSTRUCTION

COLUMN NO.

IN TRODUC

1-8

Enter any introductory comments here (unrestricted input).

1-80

zz

1-2

MOLAR CO (Molar conversion factor calculations are dealt with in this section).

1-8

PRINT (if record is required. If not, leave blank.) Enter name of chromophore. Enter wavelength of light in nm. Enter optical path in mm.

1-5 11-34 41-50 51-60

Enter reading with that concentration. Enter blank with that concentration. Enter next set of values.

11-20 21 -30 ~

Enter comments on molar conversion factor calculations. (Unrestricted input.)

1-80

ZZ (if PRlNT has not been requested above, omit comments and Z Z ) .

1-2 ~~~~

INCUBATI (Incubation time and chart speed determinations are dealt with in this section.)

1-8

PRINT (if record is required. If not, leave blank.) Enter time used to determine chart speed, in min. Enter distance between marks in chart speed determination, in cm. Enter distance between marks in incubation time determination, in min.

1-5 11-20 21-30 31-40

Enter comments on incubation time and chart speed determination (unrestricted input).

1-80

ZZ (if PRINT has not been requested above, omit comments and 22).

1-2

LlNE VOL (line volume calculations are dealt with in this section).

1-8

197

COMPUTER PROGRAM

INSTRUCT1 ON

COLUMN NO.

PRINT (if record is required. If not, leave blank.) Enter number of lines pumping liquid (I). Enter total line volume observed, in ml/min. Enter Enter Enter Enter Enter Enter

1-5 11-12 21-20

line number (I). c colour of line. expected line volume, in ml/min. reading with that line. blank with that line. next set of values.

1-2 11-18 21-30 3140 41-50

Enter comments on line volume calculations (unrestricted input).

1-80

Z Z (if PRINT has not been requested above, omit comments and Z Z ) .

1-2

LlNE REA

1-8

~

PRINT (if record is required. If not, insert a blank card.) Enter line number of components (I). Enter description of component in reaction mixture. Enter concentration of components in reaction mixture. Enter concentration of component in stock solution. Enter units of Concentration. Enter volume of line reagent required, in ml (F6.l). Enter next set of values. END (after last set of values).

1-5 1-2 1 1-34 4 1-50 51-60 6 1-68 71-76

t

~

78-80

Enter comments on line reagent and reaction mixture calculations (unrestricted input).

1-80

Z Z (if PRINT has not been requested above, omit comments and ZZ).

1-2

Enter details of component in stock solution. Enter molecular weight of component in stock solution. Enter volume of stock solution required, in ml. Enter concentration of component in stock solution. MOLAR (if concentration is in molarity) or: PER CENT (if concentration is in percent w/v).

t

1-24 3140 1-10 11-20 2 1-25 21-28

198

AUTOMATED ENZYME ASSAYS

INSTRUCTION

COLUMN NO.

Enter comments on preparation of stock solutions (unrestricted input).

1-80

ZZ (not optional).

1-2

GRADIENT (gradient calibration is dealt with in this section).

1-8

PRINT (if record is required. If not, leave blank). Enter reading with stock solution directly into analyzer. Enter blank with stock solution directly into analyzer. Enter initial blank with gradient. Enter final blank with gradient.

1-5 11-20 21-30 3140 41-50

Enter gradient readings (F8.3 repeatedly, 9.9 after last value).

1-80

Enter comments on gradient calibration (unrestricted input).

1-80

ZZ (if PRINT has not been requested above, omit comments and ZZ).

1-2

ASSAY CO (assay conditions are dealt with in this section).

1-8

Enter comments on assay conditions (unrestricted input).

1-80

ZZ (not optional).

1-2 ~~

~

PROTEIN (protein concentration is dealt with in this section. Three kinds of assay are selected by going to CONSTANT, VARIABLE OR GRADIENT.)

1-7

CONSTANT (for assays in which the protein concentration does not vary). PRINT (if record is required. If not leave blank.) Enter enzyme line number (I).

1-8 11-15 31-32

Enter details of enzyme. Enter protein concentration in enzyme line. Enter units of concentration (preferably mg/ml).

1-24 3140 4148

Enter comments on Protein Concentration (unrestricted input).

1-80

ZZ (if PRINT has not been requested above, omit comments and ZZ).

1-2

199

COMPUTER PROGRAM

COLUMN NO.

INSTRUCTION

(Go to next section.) VARIABLE (for assays in which the protein concentration in samples are different). PRINT (if record is required. If not, leave blank.) GRAPH (If graph is required. If not, leave blank.) Enter enzyme line number (I). Enter name and details of enzyme in sample. Enter protein concentration in sample, in mg/ml. Enter next set of values. END (after last set of values).

+'

1-8 11-15 21 -25 31-32 1-24 31-40

~

43-3 1

Enter comments on Protein Concentration (unrestricted input).

1-80

ZZ (If PRINT has not been requested above, omit).

1-2

(Go to next section.) GRADIENT (for assays in which the protein concentration is varied by the gradient making device). PRINT (if record is required. If not, leave blank.) GRAPH (if graph is required. If not, leave blank.) Enter enzyme line number.

1-8 11-15 21 -25 31-32

Enter name and details of enzyme in gradient making device. Enter protein concentration of undiluted enzyme. Enter units of concentration (preferably mg/ml).

1-24 3140 4148

Enter comments on protein concentration (unrestricted input).

1-80

ZZ (if PRINT has not been requested above, omit comments and ZZ).

1-2

SAMPLE P (The sample pattern is dealt with in this section). Enter number of sample components (I). Enter sample line number (I). Enter sample conponent number (I). Enter name and details of sample component. Enter concentration of component in sample. Enter units of concentration. Enter next set of values. E N D (after last set of values).

t

1-2 11-12 1-2 11-34 41-50 51-58 61-63 Subject index P. 218

200

AUTOMATED ENZYME ASSAYS

INSTRUCTION

COLUMN NO.

Enter comments on sample pattern (unrestricted input).

1-80

ZZ (not optional).

1-2

RESULTS (the results are dealt with in this section. There are alternative ways of entering results for different kinds of assay.)

1-7

NO PROTEIN (for assays in which the protein concentration was not determined) or: CONSTANT (for assays in which the protein concentration does not vary) or : VARIABLE (for assays in which the protein concentration in samples are different) or: GRADIENT (for assays in which the protein concentration is varied by the gradient making device). SAMPLE PTN (for assays in which there is a sample pattern) or: NO SAMPLE (for assays in which there is no sample pattern). GRAPH (if graph is required. If not, leave blank). Enter experiment number (alphanumeric characters). Enter date (alphanumeric characters).

1-10 1-8 1-8 1-8 1 1-20 11-19 21-25 31-38 41-48

Enter readings (F8.3 repeatedly, 9.9 after last value).

1-80

Enter blanks (F8.3 repeatedly, 9.9 after last value).

1-80

or: Enter initial blank. Enter final blank. 9.9

1-8 9-1 6 17-19 ~

Enter comments on results (unrestricted input).

1-80

ZZ (not optional).

1-2

Enter comments on graph (unrestricted input).

1-80

ZZ (ifGRAPH hasnot been requested above, omit commentsand

ZZ).

1-2

RECYCLE (if further assays required).

1-7

FINISHED (after last assay).

1-8

20 1

COMPUTER PROGRAM

IV.3. Program PROGRAM F O R G E N E R A L I Z E D E N Z V M E A U T O M A T I O N S V S T E H

................................................ .........................

-

M E A N I N G OF V A R I A B L E NAMES AMCF

I CB

cc

CCL CCML CCRM CCS

AVERAGE M O L A R C O N V E R S I O N F A C T O R BLINK CHROMDPHORU B L A N K C O N C E N T R A T I O N OF CHROMOPHORE COMPONEhT C O h C C h l R A l l O h I N L I N E CIIROMOPMORE C O N C E N T R A T l O h M A X I M U M L l h E A R ( V A L U E ) COMPOhEhT C O N C C N l R A T l O h I N R E A C T I O N MIXTJRE

ccss CWR

cns

CLN CMY CONTROL CR CRM CS CVL CVSR CVSS

OW DATE OC DUCS

OMIT 6 ILN 6V EXPTND VLV PBF OBI

OR

ORN

IT

LA LB LC LDF LN LR

LVO LVT MCF NCR NCRM NOR NL NPR NR NSC

NS OP

cc

CCEL CCELI PCRM PCRMl

I

I A BA DB DC 8CCL ICCRM SCN

ILN I R TLUO TLVT TMCS TR

ucc

UCCL

ucsc YL

DATE OATA C O N T R Q L D I S T A N C E B E T U E E N U A R K E R S FOR C H A R T S P E E D D E T E R M I N A T I O N D I S T A N C E B E T U E E N M A R K E R S FOR I N C U B A l l O N T I M E D E T E R M I N A T I O N EN7VME E N i V M i L I N E NUMBER ENZVME ( V A R l A B L E ) E X P E R I M E N T NUMBER F R A C T I O N A L L I N E VOLUME GRADIENT BLANK F I N A L GRADIENT B L I N N I N I T I A L GRADIENT R I A D I N G G R A D I E N T R E A D I N G NUMBER INCUBATION TIME L I N E ACCURACV L I N E BLANK L I N E COLOUR L I N E D l L U T l C N FACTOR L I N E NUMBER L I N E READING L I N E VOLUME O B S E R V E D L I N E VOLUMC T H E O R E T I C A L MOLAR C O N V B R S I O N F A C T O R NUMBER O F CYROMOPHORE R E A D I N Q S NUMBER O F COMPONENTS I N R E A C T I O N M I X T U R E NUMBER OF G R A D I E N T R E A D I N G S NUMBER O F L I N E S NUMBER O F P R D l l l N R E A D I N G S NUMBER O F N E A D I N G S NUMBER OF S A M P L E COMPONENTS NUMBER O F S A M P L E S O P T I C A L PATH. PROORAM CONTROL P R O T E I N CONCENTRATION I N ENZYME L I N E P R O T E I N CONCENTRATION I N ENZVME L I N E l N l T l A L L V PROTEIN CONCENTRATION I N REACTION MIXTURE PROTEIN CONCENTRATION I N REACTION MIXTURE I N I T I A L L Y LEADING RATE S P l C l F l C ACTIVITY STOCK B L A N K S A M P L E COMPONENT S A M P L E COUPONENT C O N C E N T R A T I O N I N L I N E S A M P L E COMCCNENT C O N C E N T R A T I O N I N R E A C T I O N M I X T U R E S A M P L E COMPONENT NUMBER S A M P L E L I N G NUMBER STOCU R E A D I N G T O T A L L I N E VOLUME O B S E R V E D T O T A L L I N E VOLUME T H E O R E T I C A L T I M E l E T Y E 6 N M A R K E R S FOR C H A R T S P E E D D E T E R M I N A T I O N TOTAL REACTION U N I T S O f C O N O E U T R A T I O N OF COMPONENT U N I T S O f C O N O E U T R A T I O N OF L I N E R I A B E N T U N I T S O F C O N C E N T R A T I O N O f S A M P L E COMPONENT UAVELENPTH Subject index P. 218

202 LINE NO.

AUTOMATED ENZYME ASSAYS

OCTAL AOnRESS

FORTRAN V.

nn

SOURCE PROGRAM

0200569

iEVEL

0010 OOlO

0010 5

10

0010 0014 OCP7

c

0033 0043 0047 OCh2

1

$QITE(6,200)

2

9"ALL E A D ( COHMEilT 5~100lPC 9 E A D ( 5 ~ 1 0 0 l P C CO') G O T O 13 IF(PC.NE.*MOLAR IF(PC.NE.*MOLARM O L ACO') R C O NGVOE RTO S I O13 N FACTOR C A L C U L A T I O N NOLIR CONVERSION FACTOR CALCULATION 9EAD(5.101) PC,CHR.IL,OP 9EAD(5.101) PC,CHR,UL*OP DO 4 I'l.1UO

C 0066 0114 0117 15

0143 0153 0155 0160

0172

0115 20

0200

0207 0213

3 1 1

00 4 I : l . l U O

4 3

1

6

1 1 1

7

0215

25

0216 0221 0223 0226

0231

30

I)

9

1 1

10

1

11

0240

0243 0247 0257

0262 0312 35

0344 0350 0363

12 13 C

0367

0413 40

0416

0421

IRITE(6,203lA~Cf,WL.oP,CC~N~,c~~

CALL C O M M E N T ~EAD(5,lUO) PC 00 TO 15 IFlPC.NE.'lNCUBATl') INCUBATION T l M A N D C:HART SPEED CALCUL.ATIONS ?EAD(5,103) PC,TMCS.DMCS.DMlT SHS.DMCS/TMCS IlsDMIT/CHS IF(PC.NE.'PR!NT ' ) G O T O 14 dRlTE(6,204IT~CS~DMCS~DM!~~CHSrIT

0425 0454

0460

45

CCML lFlP dRITE(6.2011 00 I1 1.1,HCR dRITE(6,202) C C I I ) ~ C R L I ) ~ C E ( l ) ~ M' IC! F)

14 15 C

0473

CALL C O M M E N T SEAO(5.100) P C IF(PC.NE.'LINE VOL') G O TO 21 L I N E V O L U H E CALCULATI

0477

0520 0523

50

55

60

0556 0560 0563 0573 0575 0600 0604 0607 0612 0615 0671

0631 0635 0645

65

1

16

1

17

1 1

1 1 1 1

0650

1

0713 0725 0740 0744

1

1 1

18

3RlTE

19

0

203

COMPUTER PROGRAM 0765 70

20

0771 1004

21 C

1010 1023

75

1016

1

1075

1

1101

1 1

1123 1133 1136

1

1115 1117 80

85

1

1111

1105

22 23

.....

24

1214 1220

25

1253

26

1237 1247 1267

27

C

1313 90

1321 1326 1334 1370

28

1375 1401

29

1414

30 C

1374

99

C A L L COMMENT R E I D ( 5 s 1 0 0 ) PC IF(PC.NE.'LINE R E * ' ) GO T O 26 L I N E RE4GENT AN0 R E A c T ~ O NM I X T U R E C A C C Y L A T I O N READ(5,lOOI PC

DO 24 3RlTE l C V S R ( I ),CVL( I ) C A L L COMMENT R E I D ( 5 r 1 0 0 ) PC IF(PC.NE.'STOCK SO') 00 T O 3 0 STOCK S O L U T I O N S Y R l T E ( 6 211) REID(5siO7lCS*CW SEIDl5,108)CVSS,CCSS.UCC,DC IFLUCC.EO.'PER C E N T ' ) CMY= 1 0 . 0 CU.CMY.C~SS.CCSS/lOOO. IF(UCC.EO.'PER CENT*)CMY=O.O 3RITE(6,212lCS,CMU,CCSS.UCCICVSS.CY IF(DC..KO.'END') 00 TO 2 9 8 0 T..O 2 7 _. C A L L COWMEN7

'.CCL

ccs I I 1 . U C t L

..

1420 1447

100

105

1452 1467 14?2 1477 1520 1525

1530

1 1 2 2

31

1

33

32

1551

110

115

1555 1565

1570 1572 1617 1625 1631 1644

1

1

34 35

J6 P

C

1650 1660

1664 1677

120

1703

125

1723 1727 1752 1716 1717

1763 1/66 2021

130

2075 2035

37 C

38

1

1

1

39

1

41

40

2041

2064

135

2067 2100 2104 2110

I.1.NOR PCRM(I).PCELI.FLVIELNl.GDF(I) DO 4 1

42

2170 2146

140

DRN.1 URlTE(6.214) GRN,GR(I),GOF(I) C A L L PLoT(NGR.GOF) C A L L COMMEIIT R E A D ( 5 , l U O ) PC IFIPC.NE.'ASSAY CO') GO TO 3 7 RESULTS ASSAY CONDITIONS dRITE(6.215) C A L L COMMENT R E A D ( 5 s I O U l PC IF(PC.NE.'PROTEIN G O T O 50 P R O T E I N CONCENTRATIONS READ(5,lllICONTROL.ELN IF~CONTROL(~~.NE.'CONSTAN G O~ ' TO ~ 38 R E I D ( S r 1 1 2 l EIPCEL~.UCC PCRMI.PCELI.FLV(ELN) DO T O 42 IFLCONTROL(l).NE.'VIRIABLE'I G O TO 4 0 00 39 1.1,200

2147 2153

43

IF(CONTROL(P).NE.'PRINT 1)GO T O 4 9 I F ( C O N T R O L ( l ) . N E . t C O ~ S T A N T I ) GO T O 4 3 YRITE(6.216) YRITE(6.217) E,PCELI.PCRMIsUCC 30 1 0 4 7 IF(CONTROL(l).NEI'VARIABLEI1 GO 70.45

2155 2165 2170

1

44

Subjecr index P. 218

204

AUTOMATED ENZYME ASSAYS

2245 2251 2?74 2277 150

155

2324

45 1

46

2316 2342 2343

47

2353

49

I F ( C O N T R O L I l ) . N E , ' G R A D I E N T ~ ) G O TO 4 4 dRITE(6.223) EIPCELI~UCC 00 46 I = l . V G R dRITEI6.221) I r G n F ( I ) s P C R I I ( I l IF(CONTROL(J).EU,'GRAP.I ' ) CALL PLOT I N O R ~ P C R M ) :ALL COHHEVT 30 TO 49

5u C

2366 2372 241 0

24j 3

160

165

170

175

2454 246@

2464 2466 2476 2501 2595 2555 2561 2574 2600 2613 2641 2644 2661 2664 2671

2712

180

2717 2722 2737 2742 2747 2770 2775

I 1

1

1

1

51

52

53 54

1 1

2 2

55 56 00 57-J*J149 SEAD15~llO)L DO 57 Isl.1U IF(ABSIZIIl).CT.5.)

1 1

2 2

57 58

190

195

3002 3004 SO07

1

59

30P1 3022

600

3n25

60

3030 3134 3052 30hl 3065 3071 3075 3101

1 1 1

3114

3172 3123 3126 3135

205

3136

610

62 63 64

3164 3170 3173

68 69 71 70

3235 215

3321

12

3315

333i

220

3337 3347 3355 3365 3371 3372

3402

00 T O 76

)

65 66 67

3231 3232

IFlN0.NE.NH)

TE ( 6 . 2 3 1 )

3141 3150

3160

210

QO T O 6C

bO T O 6 0 0

61

3111 200

00 TO 58

W=I+lU*J-l

IF(NB.NE.2)

3000 185

!J(l*lO*J)'ZII)

76 73

G U T O 616 GO TO 63 GO T O 6 5 G O T O 65

205

COMPUTER PROGRAM 225

3415 3421

74

3425 3435

230

235

240

3445 3445 3445 3415 3445 3445 3445 3445 3445 3445 3445 3445 3445

3445

3445 245

3445 3445

75

C 1CG 1111

1u2 103

1 'I 4 10 5

106 It17

lU8 109

113 111 112 113 114

115 116

C 3445

ZUO

3445

2111

IFlPC.EO.~FINISHED~lGO TO 7 5 IFlPC.EO.'SECYCLE 8 ) GO TO 1 0 0 0 ~R1TE16~2331 dRIIE16r23dI FORMAT S T A T t H E N T S I l N P U T )

FORMAT(A81 F O R M A T I A 8 ~ 2 X . 3 A 8 ~ 6 X ~ 2 F l 4J ). FORMAT(3F10.4,A3I FORVA~118.2X~3F10.4) ~ O R H A T ~ A ~ ~ ~ X I I ~ ~ ~ X ~ F ~ O . ~ J FORMATl12~RX~A8~2X~3F10.4) FOR*AT(12.BX~318,0X.ZF13~4~A8.2X,F6.l,lX,A3) FORMAT(3A8.6XsFlU.4I FOaMAT12F10.4r*U.2X.A3l FOHMATlA8,ZX,41FlU.4Jl FORMAT(~OF~.~I FORPAT(3lAar2XI~121 FORMAT13A8,6X~Fl0.4i40)

FORfiAI13A8~6X~FlO.~~A3)

FORMAT(2112~8X)l FORMAT1 12, B X , 3 A B , 6 X , F l O . I s A8,ZXs A 3 1 FORMAt15lA8s2XlI FORMAT S T A T E M E N T S 1 O U T P U T ) FORMAT1131/1ME130X,'I N T R 0 0 U C T I 0 '49 1/1301 FORMAT(lUlI1H~r30Y~'C A L I R R 4 T I 0 N*/lqE 1,2UX,'MOLA4 C O N V E R S I O N FACTOR C I L C U L A T I O N ' / / l l X , 2'CHROnOPUOdt REIOING d L A INK MO L A R C UNV t E S I ON / 1 l X 3'CONCENTRATION'27X, FACTORlUHOLES/ML'/l1X, 4'IUMOLESIMLJ'~29X.'THAT GIVE A '/11X, OF l . O * / I 541X,'READIVti

,

3445 3445

250

255

202 233

3445

204

3445

2U5

3445

2n6

6445

2117

3445

208

3445

209

3445

210

3445

211

F O R ~ A T I ~ ~ X ~ F ~ . S ~ Z I A X ~ F ~ . ~ I I ~ X I F ~ . ~ I

F O R H A T I l H ~ I . l O X . ' A R E A D I N G OF l.J I S EouIVALENT TO I1F7.3,~UrOLESl M U ~ / l l X . ' W I T H A P A T H L E N G T H OF ' , F 4 , 1 a v HH. THE 2 5 E A D I N G 5 ARE P R O P O R T I O N A L T O ' / l I X , ' C t i R G H O P H O H E CONCENTRATION U P 1 3 3',F7.3,' U*ULES/ML. / 1 H .10X,'TME CHHOMOPHORE 15 I . J A U 1 F O ~ M 4 T l l ~ a ~ 2 0 X ~ ' I N C U R ATTI MI O E ~AND CHART S P E E D C A L C U L A T I O N ' / / 111X,'THE CHAHT W 4 S HUN F O R ' , F 4 . 1 , ' M I N S . AND M O V t D t , F 4 , 1 , * CHS. L ? A R K S ~ / 1 1 X , ' F O R I N C U H A T I O N T I M E M E A S U R E M N T WERE ' , F 4 . 1 , * CH. 34PARTI/llX.*THE C H A R T SPEED UAS ' , F 6 . 3 , ' c H / M [ N + AND THE1 4.' INCUBATlOhl TIME 'aF5.2,' HlN.'I FOH3AT(lHB.ZOX.'LINE VOLUME CALCULATION*I/~H , I LINE LINE 1 CALIdRATIONFR4CTlONAL LINE OBSfSVEO 2 EXPECTED LINE'IIH NUMilER COLOUR REIUING RLANX 3 LINE 0 I L U T ION LINE LlNt ACCURACY (PER 4 ' / 1 H .46X.'VflLUHE FPcion VOLUME VULUElE 5 CENT'liH ~ 7 2 X . ' l ~ L / H l N l IMLlMlNj OF r H E O R Y I * / l FORMATIlH ~ 4 X ~ 1 2 , 5 X ~ A 8 ~ 4 X ~ F 5 . 3 ~ 5 X ~ F ~ , 3 ~ b X , F 5 ~ 3 ~ l O X ~ ~ 5 . 2 ~ 9 X , f 19X*F4.2,9XsF5.1) T O R * A T I l H O ~ ' * * . WARNING L I N E ',l2,' HAS MORE T H A N I U P E H CENT P U 2 ? P l N G ERROH ..*'J F O R M 4 1 1 l H O ~ l O X ~ ' O B S E R V E D TOTAL L l N q VOLUME I S ' r F 5 . 2 . ' ML/WIN. EXP 1 P C T E D TOTAL L I N E VOLUME 1 5 1 , F 5 . 2 , I ~ L / M I N . ' / ~ ~ X I ' O A Z ~ H VALUE V~~ I S 2 ',F5.1.' PER C t N T OF F X P E C T F C I ' I FORMATIlUI/lYB,2OX. L I N t REAOENl A N D R E A C T I O N MlXTUHE CALCULATIOY' l l / l h LINC COVPONENT I N C O N C E N T H A T I O N OF CO?,PONEN 7T I N UhllTS T O PREPeRE L I N E R E A G E k T * / l H NUMeER REAC 3710N MlXTUdE Il)LIN€12JHkACTION 13)5TOCI( OF 4 M A K E 1 A I M L . O f S T O C K U P T O IB)ML'/lH ,33<.'RtAGkNT MIXTURE I A I IBI'IJ 5 SOLUTION CONCN. FORMAT(1H , ~ X I I ~ ~ Z X ~ ~ A R ~ Z X I F ~ . ~ ~ ~ X I F ~ . ~ , ~ X . F ~ . S ~ ~ X , ~ P

l * L A T #,F5,1,'

,'

1'7.2)

i O ~ * A T l 1 U l / l H 8 , 2 0 X . ' P R E P A R A T I O N OF S O L U T I O N S ' / / 1 H ,'CUMPONCNT 3F POLtCULAR CONCENTRATION UNITS V O L U M t OF S UEICHT O F ' I ~ H , I SOLUTION WEIC OF COMPONENT OF SOLUTION HEOUIHEU 4 COHPSNtNT'/lH .24X.'COHPOIUENT I1 S T O C K S O L U T I O N CONCN. 5 1ML.J ICM)'/I F O R I . A T ( l H I l ~ 8 O X ~ ' W E l G H TOF Y € S S E L ' / l M , 3 A a . 2 X . F 6 1 1 , 8 K , f ~ 7 . 3 . 9 Y I I B r LjX.Ffl.3.' l i E l t i h T OF COMPONENT 4,F9.5/1~ ,lolX~'---------~/iH

1 STOCN

ZIOCK 3 4 T UF

260

3445

212

3465

213

3445 3445

214 215

3445

216

3445

217 218

3445

,

237Xt'SUM'l F O R H 4 T l l H 1 / 1 h B , 2 Q X , ' G H A D I E N T C A L I i l ~ A T ~ O N ~ / l H O , 1 O ~ , ~ H F A D I NR EOA D 1 1UC G R A D I E N T ' 1 1 H slOX,'IIUMBER D l L U T I O N ' l l H .3UX,'FAC 2TOR 1 / I CORHATllH ,13X,13,5X,F5.3,6X,F5.31 FORUATl1Hl/lHE~3OX,'R E 5 U L T Se/lHB.POX,'ASSAY COUD IITIWS~/~H~I FORMAT(IUB,~OXI'PROTEIN CONCENTRATIONS'//lM ,IOXI'ENZYHL 1 PROTEIN CONCENTRATION I N L J N I T S ' / l H ,33X.'EiNZYME REACT ZION O F ' I l H .34X,'LINE nlxTuRE CONCN.'//I FORnATllH ~8X.3AR.lX.F7.3~5X,F7.318X.1A) FOr7MATlIHE,20X.'PROTEI~ CONCENT~ATIONS~//1lX~'SA~PLt'~IX,'ENZYME'~ 1 1 ' X ~ ' P R O T E l N CONCN. l l l ' / l l X ~ ~ N U ~ B E R ' ~ ~ 4 X ~ ~ E N RLEIAHC tT I O N ' / I l X . 2'LINE MIXTURE'/)

Subject index P. 218

AUTOMATED ENZYME ASSAYS 265

270

275

280

3445 3445

219

22"

3445 3445 3445

221 222

3445 3445

224

3445

226

3445

227

3445

228

3445

229

3445

230

223

225

3445

231

3445

232

3445

233

3445 3445

234 235

3445 3446

LINE NO.

OCTAL ADflRESF 3453 345 3 ~. 3453 3463 3503 3 5n7 .~

3513

FUHTHAN V .

00 LEVEL

SUURCh PilUORAH

*FORTRAN SOURCE SUBROUTINE COMMENT TEXT T C , T ( P ) ~ R I T E ~ 6 ~ 2 D l l I ? E A D 1 5 , 1 d 0 1 1C.T GO TO 2 IF(TC.EO.'LISTDATA'I IF(TC.EU.'ZZ ' I G O TO 4 I F I T C . E O . ' L A S T C 4 R U ~ l G O TO 4

3517 3537 3540 3550 3510 3574

3614 3615

3615 3615 3615 3615 3616

100 200

FDRMATlAR.PA81

202 4

F O A M A T I ~ H U ~ ~ O X I ' C H E C KOF COMPUTER I N P U T ' / / ) 3ETURN IND

FDRMAT(1H , A 8 , 9 A 8 1

201

COMPUTER PROGRAM

LINE NO.

5

10

15

20

29

30

35

40

OCTAL AOnRESS

3622 3622 3622 3633 3633 3633 3651 3640 3653 3666 3671

3677 3702 3715 3727 3732 3712 3761 3771 3773 3776 4002 4005 4010 4014 4017 40'23 4026 4832 4055

4040 4017 4062 4071 4111

4111 4hil 4111 4111 4111 4112

FORTRAN V.

DO

1

1

1

2

3

C 1 1 1 1

1 1

1

-.... ..

0200569

II

-.

lF(NX.Ll.251

1

1 1 1

IX?XI

00 3 I Z 1 , N X

1

SUURCO PROGRAH

WRITE(6.2031

LINEfJ).SYMEOL WRITE(6,2011 1,LlNE LINE(J).DOT #RITE(br2021 INSERTION OFSCALE DIVISIONS LINE(IUI=EIR 00 4 1=1.10

4 9

6 7

1

1 1

0 9

20u 201 202 205 204

Subject index

p . 218

References

ABDULLAH, M. I. (1967) In: Automation in Analytical Chemistry, (Mediad Press, New York) Vol. 11, pp. 401-405 ABOU-DONIA, M. B. and D. B. MENZEL (1967) Comp. Biochem. Physiol. 22,99 ALLEN,E. (1966) Clin. Chem. 12, 376 ANDERSON, N. G. (1969) Anal. Biochem. 28, 545 F., D. MAGNANELLI and V. BOFFI(1967) In: Automation in Analytical AVANZINI, Chemistry (Mediad Press, New York) Vol. 11, pp. 285-290 H. B. EKMAN, and D. KNUTSSON (1965) In: Automation in Analytical AXELSSON, Chemistry (Mediad Press, New York) pp. 603-608 and S. HUNT(1967) Science Tools (LKB Instrument AYAD,S. R., R. W. BONSALL Journal) 14, 40 H., K. S. CHIOand A. L. TAPPEL(1969) Anal. Biochem. 29,515 BARRERA, (1965) In: Automation in Analytical Chemistry BARRON, E. J. and A. BOULEY (Mediad Press, New York) pp. 359-362 (1967) Anal. Biochem. 21,208 BECK,C. and A. L. TAPPEL H. and G. PALMER (1965) Advan. Enzymol. 27,105. BEINERT, BERGMEYER, H.-U. (ed.) (1968) Methods of Enzymatic Analysis (Academic Press, New York and London) S. A. (1968) Structure and Function of Enzymes (Benjamin, New York) BERNHARD, J. J., N. VIDONand F. MAISON(1966) In: Automation in Analytical BERNIER, Chemistry (Mediad Press, New York) Vol. 11, pp. 289-292 BERRY,M. N. and A. K. WALLI(1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. IT, pp. 389-393 Bibliography of Automated Enzyme Research (1965) Technicon Instrunients Corporation, Chauncey, New York BIGLEY,R. H., P. STENZEL, R. J. JONES,J. 0. CAMPOS and R. D. KOLER(1968) Enzymol. Biol. Clin. 9, 10 F. C. NEALE,S. POSENand J. BRUDENELL-WOODS BIRKETT, D. J,, R. A. J. CONYERS, (1967) Arch. Biochem. Biophys. 121,470 208

REFERENCES

209

BODANSKY, 0. (1953) J. Biol. Chem. 205, 731 0. (1959) Amer. J. Med. 27, 861 BODANSKY, BODANSKY, 0. and M. K. SCHWARTZ (1961) In: J. H. QUASTEL (ed.) Methods in Medical Research 9, 79 BORNER, K. (1968) Z. Klin. Chem. 6,269 (1967) Clin. Chem. 13, 1024 BRIGGS,A. R. and H. V. CONNERTY BRILL,A. S., A. EHRENBERG and H. DENHARTOG (1960) Biochim. Biophys. Acta 40, 313 BROOKS,L. and H. G. OLKEN(1965) Clin. Chem. 11,748 BROWN,H. H. and M. R. EBNER (1967) Clin. Chem. 13,847 BUCKLEY, R. (1969) Process Biochem. 4, 67 BURNS,D. A. and L. LAZER(1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 4 2 4 and J. KACSER (1965) J. Chromatography 20,310 BURNS,J. A,, C. F. CURTIS S. (1966) In: Automation in Analytical Chemistry (Mediad Press, New BURROWS, York) Vol. I pp. 462-464 CADMUS, M. C. (1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 556-657 CAMERON, C. B. and N. W. WEG(1964) Technicon Symposium London (reprint) Technicon Instrument Co., Chertsey, Surrey (1966) In: Automation in Analytical Chemistry CAMIER,M. and P. GONNARD (Mediad Press, New York) Vol. 11, pp. 367-370 CAMIER, M. and P. GONNARD (1968) Bull. SOC.Chini. Biol. 50, 933 R. 0. BRIEREand R. R. L. CALAM (1966) Clin. CAPPS,R. D., H. G. BATSAKIS, Chem. 12,406 CATRAVAS, G. N. and E. D. LASH(1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. 1, pp. 4 4 - 6 CHANCE, B. (1951) Adv. Enzymol. 12, 153 CHANEY,A. L. (1967) In: Automation in Analytical Chemistry (Mediad Press,New York) Vol. I, pp. 115-1 I7 (1966) In: Automation in Analytical CHARIOT,J., C. ROZEand M. SOUCHARD Chemistry Mediad Press, New York) Vol. 11, pp. 383-388 CHEN,R.F., A. N. SCHECHTER and R. L. BERGER (1969) Anal. Biochem. 29,68 (1967) In: Automation in Analytical CHERSI,A., P. G. PIETTAand A. CORBELLINI Chemistry (Mediad Press, New York) Vol. 11, pp. 223-226 D. D. and R. NICKLAS(1966) In: Automation in Analytical Chemistry CLARKE, (Mediad Press, New York) Vol. I , pp. 465-8 CLELAND, W. W. (1963) Nature 198, 463 CLELAND, W. W. (1967) Advan. Enzymol. 29, 1 COLEMAN, C. M. and R. C. STROJE (IS65) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 575-577 S. P. and N. 0.KAPLAN (eds.) (1955 onwards) Methods in Enzymology COLOWICK,

210

AUTOMATED ENZYME ASSAYS

(Academic Press, New York) (1966) Clin. Chim. Acta 14,263 COMFORT, D. and D. 5. CAMPBELL COOKE, K. B. and 5. V. PATSTON (1961) Proc. Assoc. Clin. Biochemists I, 150 and J. J. TRIAS(1967) Clin. Chem. COTLOVE, E.,D. S. YOUNG,0. W. MORTON (abstracts) 13, 678 CRAMP, D. G. (1967) J. Clin. Pathol. 20, 910 CRAMP,D. G. (1968) 5. Clin. Pathol. 21, 171 DAVIS,G. A., R. J. SANTEN and B. W. AGRANOFF (1965) Anal. Biochem. 11, 153 DIXON,M. (1951) Manometric Methods as applied to the Measurement of cell Respiration and other Processes, (Cambridge University Press, Cambridge) DIXON, M. and E. C. WEBB(1964) Enzymes, 2nd edition (Longman, Green and Co., London) and J. A. KNIGHT(1968) Amer. J. Clin. Pathol. 50, 491 DUBE,V., M. D. HUNTER DUGGAN, E. L. and N. GURLL(1965) Anal. Biochem. 11, 281 (1957) Biochim. Biophys. Acta 24,19 DWSENS,L. N. M. and J. AMESZ DYSON, J. E. and E. A. NOLTMANN (1965) Anal. Biochem. 11,362 EIGEN,M. (1968) Quart. Rev. Biophys. I, 3 (1963) Advan. Enzymol. 25, 1 EIGEN,M. and G. G. HAMMES Enzyme Nomenclature (1965) Elsevier Publishing Co., Amsterdam (1960) Federation Proc. 19, 39 ESTABROOK, R. W. and H. GUTFREUND EVENSON, M. A., G. P. HICKS,J. A. KEENAN and F. C. LARSON (1967) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 137-146 (1963) J. Clin. Pzthol. 16, 170 EVERARD, D. and G. SEYMOUR T. P. WHITEHEAD and G. M. WIDDOWSON (1966) In: FARR,R. F., J. A. NEWELL, Automation in Analytical Chemistry (Mediad Press New York) Vol. 11, pp. 225-229 FINGERHUT, B., R. FERZOLA, H. W. MARSHand J. B. LEVINE (1962) Ann. N. Y. Acad. Sci. 102, 137 (1967) Advan. Clin. Chem. (eds. 0. BODANSKY FISHMAN, W. H. and N. K. GOSH and C. P. STEWART) 10, 255 FISKE,C. T. and Y. SUBBAROW (1925) J. Biol. Chem. 66, 375 FLEISHER, G. R. (1967) Clin. Chem. 13, 233 FURNESS, F. N. (ed.) (1960) Ann. N.Y. Acad. Sci. 87, 609 GAGE,J. C. and M. H. LITCHFIELD (1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. 11, pp. 395-100 and R. R. SCHAFFERT (1964) Clin. Chem. 10, 540 GETCHELL, G., G. R. KINGSLEY Q. H. and L. MILNES(1964) Biochem. J. 91, 161 GIBSON, GIRARD,M. L., F. ROUSSELET and M. KOCH(1963) Ann. Biol. Clin. (Paris) 21, 557 M. L., J. CANAL and J. JOUANNELLE (1966a) In: Automation in Analytical GIRARD, Chemistry (Mediad Press, New York) Vol. 11, pp. 375-381 and J. JOUANNELLE (1966b) Ann. Biol. Clin. (Paris) 24, GIRARD,M. L., J. CANAL 1191

REFERENCES

21 1

GIRARD, M. L., J. CANALand J. JOUANNELLE (19664 Ann. Biol. Clin. (Paris) 24, 1205 GOULD,H. E. (1967) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 141-146 GREEN,S., T. J. GIOVANNIELLO, R. A. COTBand W. H. FISHMAN (1467) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 563-570 GREEN,S., T. 5. GIOVANNIELLO and W. H. FISHMAN (1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 480488 GROFF,W. A., L. A. MOUNTER and V. M. SIM(1966) In:Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 498-502 Guide to Scientific Instruments (1968) Science 162A GUTFREUND, H. (1965) An Introduction to the Study of Enzymes (Blackwell, Oxford) GUTFREUND, H. and B. R. HAMMOND (1963) Nature 198, 667 HARRISON, N. B. (1966) Clin. Chem. 12, 890 HARVEY, R. A. (1969) Anal. Biochem. 29, 58 HAZEN,G. G., J. A. HAUSEand J. A. HUBICKI (1965) Ann. N.Y. Acad. Sci. 130, 76 1 0. LEVAND and M. MCCARTER (1967) In:Automation HEINICKE,R. M.,C. LARSON, in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 207-212 HENRY, J. B., E. S. CESTARIC and A. GOODWIN (1963) Amer. J. Clin. Pathol. 40,252 HICKS,G. P. and S. J. UPDIKE(1965) Anal. Biochem. 10, 290 (1966) Anal. Biochem. 16, 327 HILL, J. B. and D. S. COWART HILL,J. B. and G. KESSLER (1961) J. Lab. Clin. Med. 57, 970 HILL,P. G. and H. G. SAMMONS (1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 601-602 (1966) Clin. Chim. Acta 13,739 HILL,P. G. and H. G. SAMMONS HILL,H. H., G. K. SUMMER and M. D. WATERS (1968) Anal. Biochem. 24,9 HOCHELLA, N. J. and S. WEINHOUSE (1965a) In:Automationin Analyticalchemistry (Mediad Press, New York) pp. 539-544 HOCHELLA, N. J. and S. WEINHOUSE (1965b) Anal. Biochem. 13, 322 HOCHELLA, N. 5. and S. WEINHOUSE (1965~)Anal. Biochem. 10, 304 (1964) Anal. Biochem. 9,467 HOOBER, J. K. and I. A. BERNSTEIN HOPKINSON, D. A. and W. H. P. LEWIS(1967) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. 11, pp. 227-231 HORNE,M., C. J. CORNISH and S. POSEN(1968) J. Lab. Clin. Med. 72,905 HOUGHTON, B. J. (1964) Reprint No. 47, Technicon 25th Anniversary International Symposium, London C. G. and G. J. WRIGHT(1967) Toxicol. Appl. Pharmacol. 10,467 HUMISTON, HVIID,K. (1967) Clin. Chem. 13, 281 ILLINGWORTH, J. A. and K. F. TIPTON(1969) Biochem. J. 115, 51 1 JOHANSEN, G. and R. LUMRY (1961) Compt. Rend. Trav. Lab. Carlsberg 32, I85

212

AUTOMATED ENZYME ASSAYS

JOLLES, P., M. BONNAFB, H. MOUTON and L. SCHWARZENBERG (1967)Rev. Franc. Etud. Clin. Biol. 12,996 JONNARD, R. (1960)Ann. N.Y. Acad. Sci. 87,669 JOWETT,G. K. and D. B. ROODYN (1969)Technicon Symposium London (in press) JUNGNER, I. and G. JUNGNER (1968) In: Symposium on Multiple Diagnostic Screening Analysis (University of Minnesota) May 1968 KALDOR, J. and D. J. SCHIAVONE (1968)Clin. Chern. 14,735 KAUPPINEN, V. and C. G. GREF(1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. 11, pp. 401-405 KAWERAU, E. (1966)Z. Klin. Chem. 4,224 KEAY,H. and J. A. TREW(1964) Clin. Chem. I0,75 KINDIG,W., L. H. NIELSON and S. R. INMAN (1967)Clin. Chem. (Abstracts) 13, 678 KING,E. J. and D. M. CAMPBELL (1960)Clin. Chirn. Acta 6,301 KIRK,D. L. (1967)In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 559-562 (1966)Clin. Chem. 12,289 KLEIN,B. and J. AUERBACH KLEIN,B. and 5. H. KAUFMAN (1967)Clin. Chem. 13,290 KLEIN,B., J. AUERBACH and S. MORGENSTERN (1965)Clin. Chem. 11,998 KLEIN,B., M. OKLANDER and S. MORGENSTERN (1966)Clin. Chem. 12,226 KLINE,M. H., F. MEDZIHRADSKY and L. E. HOKIN(1968)Anal. Biochem. 23, 97 KOWALSKY, A. and M. COHN(1964) Ann. Rev. Biochem. 33,481 LAMY,J. N., J. LAMY-PROVENSAL, J. DE RUSSEand 1. D. WEILL(1967)Bull. SOC. Chim. biol. 49, 1167 LECLERC, H. (1967)Ann. Inst. Pasteur (Paris) 113,713 LEIGHTON, F., B. POOLE,H. BEAUFAY, P. BAUDHUIN, J. W. COFFEY, S. FOWLER and c. DE DUVE(1968)J. cell. Biol. 37,482 J., S. L. JOHNSON, R. W. HYMAN and G. P. H~ss(l965)Anal.Biochem.II, LENARD, 30 LEVINE, J. B. and J. B. HILL (1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 569-574 and V. A. NELSON (1965)1n:AutornationinAnalytical LEVINE, J. B., R. A. SCHEIDT Chemistry (Mediad Press, New York) pp. 582-585 LEVY,A.L., C. DALMASSO and J. DALY (1965)In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 551-554 (1965)Clin. Chern. 11,367 LOGAN,J. E. and D. E. HAIGHT LOKEN,H. F. (1966)Scand. J. Clin. Lab. Invest. 18 suppl., 92,99 LUNDEGARDH, H. (1959)Endeavour 18, 191 LYSIONOK, A. A. and T. G. FLYNN (1969)Lab. Practice 18,1066 MALTBY, D. H. (1968)Bio-Medical Engineering 3,509 MARRACK, D.and A. HALL(1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 578-591

REFERENCES

213

MARSH,W., B. FINGERHUT and E. KIRSCH(1959) Clin. Chem. 5,119 R. W. (1960) Anal. Biochem. 2,141 MCGILVERY, MCKAY,D. K., D. SELIGSON and W. R. JEWETT (1964) Clin. Chem. 10,907 Y.,J. FORICHON and C. STUDIEVIC (1966) In: Automation in Analytical MINAIRE, Chemistry (Mediad Press, New York) Vol. I1 pp. 145-150 (1967) Brit. J. Haematol. I3 (suppl.), 54 MIWA,S., M. KANAIand S. NOMOTO MOORE,J. J. and S . M. SAX(1969) Clin. Chern. 15,730 MORGENSTERN, S., R. FLOR,G. KESSLER and B. KLEIN(1965a) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 545-550 S., R. FLOR,G. KESSLER and B. KLEIN(1965b) Anal. Biochem. 13, MORGENSTERN, 149 MORGENSTERN, S., G. KESSLER, J. AUERBACH, R. V. FLOR and B. KLEIN(1965~) Clin. Chem. 11, 876 S., G. KESSLER,J. AUERBACH, R. V. FLOR and B. KLEIN(1965d) MORGENSTERN, Clin. Chem. 11, 889 MORGENSTERN, S., R. FLOR, G. KESSLER and B. KLEIN(1966a) Clin. Chem. 12,274 S., M. OKLANDER, J. AUERBACH, J. KAUFMAN and B. KLEIN(1966b) MORGENSTERN, Clin. Chem. 12, 95-1 1 1 MORGENSTERN, S., J. KAUFMAN and B. KLEIN(1967) Clin. Chem. 13,270 MUNDRY, K. W. (1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 612-614 (1967) In: NASH,F. D., M. D. BAILIE,R. L. WATHENand H. H. ROSTORFER Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 599-602 (1967) Clin. Chem. (Abstracts) 13, 677 NEESE,J. W. and A. MATHER J. B. and M. D. CANNON (1955) Anal. Chem. 27,29 NEILANDS, NOTHSTEIN, D. L. and L. D. ELLERBROOK (1962) Am. J. Clin. Pathol. 37,104 M. and J. DORCHE (1968) Clin. Chim. Acta22,363 NYSSEN, OPHER,A. W., C. S. COLLIER and J. M. MILLER(1966) Clin. Chem. 12,308 S. and W. GENNARO (1966) Am. J. Clin. Pathol46,69 PASSEN, PATIENT,D. A. (1960) Ann. N. Y. Acad Sci. 87, 830 L. C. (1968) Brit. Med. Bull. 24, 189 PAYNE, PETERSON, E. A. and H. A. SOBER (1959) Anal. Chem. 31, 857 and TH. BUCHER(1962) Biochem. Biophys. Res. PETTE,D., M. KLINGENBERG Commun. 7,425 PITT,W. W. and C. D. SCOTT(1969) Anal. Biochem. 30,58 PITOT,H. C. and N. PRIES(1964) Anal. Biochem. 9,454 and A. CUTLER (1965) In: Automation in AnaPITOT,H. C., N. PRIES,M. POIRIER lytical Chemistry (Mediad Press, New York) pp. 555-558. PITOT,H. C., M. POIRIER, A. CUTLER and C. SHANNON (1966) In: Automation in Analytical Chemistry (Mediad Press, New York,) Vol. I, pp. 494-497 PITOT,H. C., M. WRATTEN and M. POIRIER (1968) Anal. Biochem. 22,359

214

AUTOMATED ENZYME ASSAYS

POLLARD, A. (1964) (abstract No. 52) Technicon 25th Anniversary Symposium. London C. J. CORNISH and F. C. NEALE(1967) POSEN,S., D. J. BIRKETT,R. A. J. CONYERS, In: Automation in Analytical Chemistry (Mediad Press, New York,) Vol. I, pp. 583-585 PRESSMAN, B. (1967) Methods Enzymol. 10,714 J. H. (ed.) (1961) Methods Med. Res. 9, 1 QUASTEL, RADIN,N. (196T) Clin. Chem. 13,55 RAIKHMAN, L. M. (1966) Biokhimiya 31,277 and N. GOCHMAN (1966) In: Automation in RATLIFF,C. R., G. S. THRASHER Analytical Chemistry (Mediad Press, New York,) Vol. I, pp. 477-479 REED, D. J. (1968) In: Atomlight, New England Nuclear Corporation, Mass., USA NO. 66 pp. 1-7 REID,R. H. P. and L. WISE (1967) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. 11, pp. 159-165 ROBIN, M. and A. SAIFFER (1965) Clin. Chem. 11, 840 ROBINSON, R., T. J. BONHAM, G. POXON and T. KELLEHER (1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. 11, pp. 211-214 D. B. (1964) Technicon International Symposium, London (reprint) ROODYN, D. B. (1965a) Int. Rev. Cytol. 18, 99 ROODYN, ROODYN, D. B. (1965b) Nature 206,1226 D. B. (1965~)In: Automation in Analytical Chemistry (Mediad Press, ROODYN, New York) pp. 593-594 D. B. (1967a) Nature 216, 1033 ROODYN, D. B. (1967b) In: Automation in Analytical Chemistry (Mediad Press, ROODYN, New York) Vol. 11, pp. 233-237 ROODYN, D. B. (1969) Process Biochem. 4, 57 D. B. and L. A. GRIVELL (1968) FEBS Letters I, 166 ROODYN, D. B. and L. A. GRIVELL(1969) Proc. Vth. Meeting FEBS L. ERNSTER, ROODYN, (ed.) Mitochondria, Structure and Function (Academic Press, London) Vol. 17 ROODYN, D. B. and N. G. MAROUDAS (1968) Anal. Biochem. 24,496 ROODYN,D. B. and D. WILKIE(1967) Biochem. 5. 103, 3c ROODYN,D. B., J. W. S m and T. S. WORK(1962) Biochem. J. 83,29 ROOS,K. (1965) Scan. J. Clin. Lab. Invest. 17, 197 Roos, K. (1966) Clin. Chim. Acta 13,403 J. W., K. J. PFAFF,F. J. SERVICE, G. D. MOLNARand E. ACKERMAN ROSEVAER, (1969) Clin. Chem. 15, 680 SAIFER, A. and M. ROBIN (1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 589-592 SCHAFFERT, R. R., G. R. KINGSLEY and H. GETCHELL (1964) Clin. Chem. 10, 519 SCHAIBERGER, G. E. and A. FERRARI (1960) Ann. N.Y. Acad. Sci. 87,890

REFERENCES

215

SCHEIDT, R. A. (1964) Technicon International Symposium, New York (Reprint No. 61) SCHEIDT, R. A., V. A. NELSON and J. B. LEVINE (1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 563-568 SCHERSTEN, B. and G. TIBBLING (1968) Clin. Chem. 14,243 0. (1965) Anal. Biochem. 13, 475 SCHEURBRANDT, SCHOFIELD, C. F. (1967) A Manual of the Atlas FORTRAN V Language (University of London Atlas Computing Service, London) SCHUEL, H. and N. G. ANDERSON (1964) J. Cell Biol. 21, 309 SCHUEL, H., S. R. TIPTONand N. G . ANDERSON(1964) J. Cell Biol. 22,317 SCHWARTZ, M. K. (1967) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 587-592 SCHWARTZ, M. K. and 0. BODANSKY (1963) In: D. GLICK(ed.) Methods of Biochemical Analysis (Interscience, New York) Vol. 11, pp. 211-246. (1966) In: Automation in Analytical SCHWARTZ, M. K. and 0. BODANSKY Chemistry (Mediad Press, New York) Vol. I, pp. 489493 SCHWARTZ, M. K. and 0. BODANSKY (1968) In: D. GLICK(ed.) Methods of Biochemical Analysis (Interscience, New York) Vol. 16, pp. 183-218 (1960a) Amer. J. clin. Path. 33, SCHWARTZ M. K., G. KESSLERand 0. BODANSKY 275 SCHWARTZ, M. K., G. KESSLER and 0. BODANSKY (1960b) Ann. N.Y. Acad. Sci. 87,616 and 0. BODANSKY (1961) J. Biol. Chem. 236,1207 SCHWARTZ, M. K., G. KESSLER D. M., A. A. STEIN,E. A. MENEGAUX, M. A. GALLOand F. COULSTON SERRONE, (1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 586-588 SEVERINGHAUS, J. W. and A. F. BRADLEY (1958) J. Appl. Physiol. 13, 515 SHULMAN, R. G. (1962) Ann. Rev. Phys. Chem. 13,325 SIEGEL,A. L. and P. S. COHEN(1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 474-476 SKEWS,L. T. (1957) Am. 5. Clin. Path. 28, 311 (1964) Clin. Chem. 10,918 SKEGGS, L. T. and H. HOCHSTRASSER SKEWS,L. T., K. E. LENTZ,J. R. KAHNand H. HOCHSTRASSER (1968) J. exp. Med. 128, 13 SLATER, E. C. (1966) In: J. M. TAGER, S. PAPA,E. QUAGLIARIELLO and E. C. SLATER (eds.) Regulation of MetabolicProcesses in Mitochondria(E1sevierAmsterdam) pp. 166178 S. MORGENSTERN and L. T. SKEGGS (1967) In: SMYTHE,W. J., M. H. SHAMOS, Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 105-1 13 (1966) In: Automation in Analytical Chemistry (Mediad SORIN,B. and R. VARGUES Press, New York) Vol. 11, pp. 469482

216

AUTOMATED ENZYME ASSAYS

STEIN,H. H. and G. J. LEWIS(1966) Anal. Biochem. I S , 481 and W. R. RODERICK (1965) Ann. N. Y. Acad. Sci. STEIN,H. H., A. J. GLASKY 130,751 (1964) Clin. STERLING, R. E., A. A. WILCOX,A. G. WAREand M. K. UMEHARA Chem. 10,1112 ST EVENS,^. O., H. E. SAUBERLICH and J. L. LONG(1967) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 553-558 STRANJORD, P. E. and K. J. CLAYSON (1966a) J. Lab. Clin. Med. 67,131 6 b )Clin. Med. 67,154 STRANJORD, P. E. and K. J. C ~ ~ ~ s o ~ ( 1 9J.6Lab. STRUMEYER, D. H. and A. T. ROMANO (1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 469-473 SYED,R. A. (1967) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 571-577 SYED,R. A. (1968) Clin. Biochem. 9, 135 (1968) Amer. J. Clin. Path. 49,613 TAMMES, A. R. and C. D. NORDSCHOW TAPPEL,A. L. (1964) Technicon International Symposium, New York, Reprint No. 32 TAPPEL,A. L. (1968) Anal. Biochem. 23, 466 TAPPEL,A. L. and C. BECK(1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 559-562 TAPPEL, A. L. and C. BECK(1967) In: Automation in Analytical chemistry (Mediad Press, New York) Vol. I, pp. 593-695. Technicon Autoanalyzer Bibliography 1957/1967,Technicon Corporation, Ardsley New York, USA (1968) Techniques in Amino Acid Analysis (1966), Technicon Instruments co., Ltd., Chertsey, England (1964) Clin. Chem. 10, 246 THIERS,R. E. and K. M. OGLSEBY THIERS, R. E., R. R. COLEand W. J. KIRSCH(1967)Clin:Chem. 13,451 THIERS,R. E., W. J. KIRSCHand R. R. COLE(1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 3 7 4 D. A. and D. BOULTER (1966) In: Techniques in Amino Acid Analysis THURMAN, Technicon Instrument Co., Ltd, Chertsey, England pp. 28-35 TIETZ,N. W. and A. GREEN (1964) Clin. Chim. Acta 9,392 TRINDER, P. and J. F. KIRKLAND (1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 609-611 S. (1962) Fluorescence Assay in Biology and Medicine (Academic UDENFRIEND, Press, New York) VANDEPVBosscm, H. (1965) Clin. Chim. Acta 12,601 VANDYKE,K. and C. SZUSTKIEWICZ (1969) Clin. Cheni. 15,154 VATIER,J., A. M. CHERETand S. BONFILS(1966) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. 11, pp. 371-374 WACKER, W. E. C. and T. L. COOMBS (1969) Ann. Rev. Biochem. 38,539

REFERENCES

217

WHITBY,L. G., F. L. MITCHELL and D. W. Moss (1967) Adv. Clin. Chem. 10, 65 WHITE,L. M. and M. A. GAUGER (1968) Anal. Biochem. 23,355 WHITEHEAD, E. C. (1965) In: Automation in Analytical Chemistry (Mediad Press, New York) pp. 437451 WIENER, N. (1960) Science 131,1355 WILDING,P. (1963) Clin. Chim. Acta 8, 918 WILLIS,C. E., T. NOSALand J. W. KING(1967) In: Automation in Analytical Chemistry (Mediad Press, New York) Vol. I, pp. 579-582 WILSON,B. W. (1966) Clin. Chem. 12, 360 WILSON, S. S., R. A GUILLAN and E. V. HOCKER (1967) Amer. J. Clin. Path. 48,524 WINTER,G. D. (1960a) Ann. N.Y. Acad. Sci. 87, 629 WINTER, G. D. (1960b) Ann. N.Y. Acad. Sci. 87,875 WOOD,W. A. and S. R. GILFORD (1961a) Anal. Biochem. 2,589 WOOD,W. A. and S. R. GILFORD (1961b) Anal. Biochem. 2,601 WOOTON, I. D. P. (1968) Brit. Med. Bull. 24,219

Subject index

(Note: This index includes enzymes cited in the text but does not include enzymes that are mentioned only in Appendix I) AC Automatic Analytical System 181 acceptor stream 173 acid phosphatase 85 addresses of firms 191 AGA multi-channel system 99 alkaline phosphatase 11, 99, 105 amidase 34 Aminco-Bowman spectrophotofluorimeter 187 Aminco-Chance dual-wavelength spectrophotometer 32, 184 ASTRA Enzyme Assayer 177 ATPase 103 Autochemist 179 Autodilutor 189 617 Automatic Analyser 181 automatic pipettes 23 automation, definition of automated assay 173 future prospects 165 ff. stages in enzyme assay 23 autosensor 173 Autospenser 189 Biological Oxygen Monitor 188 blanks 159

BTL Analmatic System 181 CE 404 Colorimeter System 180 chart readers 116 chart speed, example of measurement 155 in computer program 150 cholinesterase 82, 99 clinical assays 11 Clinomak System 180 ‘closing the loop’ 23 colorimeters 182 computer program for generalized system 149 ff,, 193 ff. computers, in data processing 117 continuous-flow methods 24, 35 Data loggers 117 data processing 114 de-bubbling 43 dialyzer, Technicon 38, 54 digital output 116 diluters 189 dilution factors 41, 173 discrete-sample analyzers 24, 63 ff., 173,180 ff.

218

SUBJECT INDEX

dispensers 189 D( -)-lactate dehydrogenase 91,96,111 dual-channel systems 76, 87, 98 Eadie plot 104 electrodes, in enzyme assays 27 electrophoresis 28 Enzymat 177 enzyme assays, calculations 114 ff., 138 calibration of instruments 118 data processing 114 gradient systems 126 incubation time 122 line reagents 125 line-volume calculations 124 molar conversion factors 119 protein concentrations 131 sample pattern 131 sequence of events 14 theory of sampling 132 ff. Enzyme Analysis System (E.A.S.) 177 enzyme characterization, Michaelis constant 102 ff. multi-parameter studies 108 ff. pH optimum 106 stability 107 substrate specificity 102 temperature optimum 108 enzyme gradients 96 enzyme kinetics, in automation 13 enzyme patterns 92 enzyme units, definition 19 Eskalab Clinical Chemistry System 186 Fall curve, in sampling 132, 174 filter, Technicon 38, 5 5 flow cells 119 fluorescence measurements in enzyme assays 27 fractional line volume (FLV) 41, 42, 144, 174

219

Gas-segmentation 44 GEMSAEC System 190 generalized enzyme automation system 140 ff. Gilford 2000 multiple-sample absorbancerecorder 27, 179, 183 Gilson respirometer 32, 188 Gilson Oxygraph 187 glucose-6-phosphate dehydrogenase 82 p-glucuronidase 106 glutamate/oxaloacetate transaminase 99 glutmotransferase 66 gradients 126 ff., 145,150,156 Grant-Linson Autolab 180 Griffin-Eel Bioanalyst 180 Half-wash time (Wd)136, 174 heating bath, Technicon 39 Incubation time 71, 122, 143 input instructions, for computer program 193 interrupted-flow methods 24,59 ff., 174 Jet-mixers 44

Ki,automatic determination 105 Kintrac VII 31, 182 K,. see Michaelis constant Lactate dehydrogenase 82, 99 Lambert and Beer law 120 LINC computer 117 line dilution factor (LDF) 42 line reagents 124, 150, 155 line volumes 124, 150, 155 Lineweaver-Burke plot 104, 105 LKB High Speed Absorptiometer 187 LKJ3 Microcalorimetry System 188 LKB Reaction Rate Analyzer 178 L(+)-Iactate dehydrogenase 75, 85, 91 ff., 96, 98, 104, 109

220

AUTOMATED ENZYME ASSAYS

Longator recorder 188 lysosomal enzymes 97 Malate dehydrogenase 29, 103 manifold 37, 174 manometric systems 188 Mecolab 177, 180 Michaelis constant (Km)53, 102 ff., 139 microcentrifuge 181 molar conversion factor (MCF) 69, 119,149,155,174 monitoring of enzyme activity 81 Multichannel 300 179 multi-channel analyzers 98 ff. multi-parameter assays 33, 111 ff. mu1t iple-enzyme analysis (M.E.A .), examples 89 phosphatases 57 principles 87 ff.

NADH dehydrogenase 91 ff., 98, 111 NADPH dehydrogenase 98 Operator accessibility 165 order of reaction 14 oxidases 90 oxygen electrode 27, 33 Peak recognition 117 peptidase 34 Perkin-Elmer spectrophotometers 185 p H optimum, automatic determination 106 phosphatases 27, 57,90, 99 phosphoglucomutase 51, 57, 82 phosphohexose isomerase 57 physical methods of enzyme assay 28 pipettes, automatic 23 polynucleotide phosphorylase 57 progress curve 13, 76 ff. proportioning pump 36, 124 protein,

assay 20,83 concentration, and enzyme assays 90 calculations of specific activity 131 computer program 151 example of assay 159 interference in enzyme assay 54 pumps 189 pump tubing 36, 49 Radioactive assays for enzymes 28 reagents 148, 190 recording Warburg manometers 32 ribonuclease 82 rise curve, in sampling 132, 175 Robot Chemist 181 Sample carry-over 135, 175 sample pattern, computer program 151 example 160 in generalized system 144 sample preparation unit 189 samplers, Technicon 37 with spectrophotometers 31 sampling, and gas-segmentation 44 conservation ofexpensive reagents 51 effect of enzyme concentration 52 effect of substrate concentration 53 enzyme 50 theory 131 units 189 semi-automatic enzyme assays 26 ff., 29 ff. sensors, in enzyme assays 37 SGOT 99 single-enzyme analysis (S.E.A.) 18, 68 ff. 9

SMA/l2 multi-channel system 99, 118 SP 3000 183

SUBJECT INDEX

spectrophotometry 33, 182 steady state, in sampling 132 stock solutions 150, 155 stopped-flow systems 34, 184 substrate concentration in multi-parameter plot 112 re-cycling 95 specificity 102 succinatedeydrogenase 85,91 ff., 96,98 Technicon system 35, 178 temperature, effect on retention time 74 optimum 108

22 1

terminology 13, 54, 173 ff. tetrazolium red, in assay of enzymes 82 transferator 189 transmission (%T) 121 Units, in enzyme assays 69 Vitraton Digital Colorimeter t 86

W,, see ‘half-wash time’ washing efficiency 135, 175 work-simplified methods 11 Zonal ultracentrifuge, enzyme monitoring 84, 86 Zymat 340 177

This Page Intentionally Left Blank