Multiple enzyme analysis with computer processing of data

ANALYTICALBIOCEEMIS~Y~~,

Multiple

496-505 (1968

Enzyme Analysis with Processing of Data

I. An Automatic Method for Determination between Enzyme Activity and Protein

D. B. ROODYN

AND

Computer of the Relation Concentration

N. G. MAROUDAS

Departmentsof Biochemistry

and Botany, University College London, Gower Street, London, W.C..t., England

Received October 30, 1967

It is usual prache to determine the “initial rate” of enzyme-catalyzed reactions, i.e., to perform the assay under conditions in which the extent of reaction is proportional to time. Although it is equally desirable to work under conditions in which the reaction rate is proportional to enzyme concentration, it is generally less easy to establish that these conditions have been obtained for each assay. Many enzyme assays are now carried out in recording spectrophotometers that automatically trace the progress curve, and hence reveal any deviation from linearity. It is usually necessary to repeat the assay at several protein concentrations to establish that the initial rate is indeed proportional to enzyme concentration. Since it is rather tedious to do this manually, we have made a simple adaptation of the Technicon AutoAnalyzer so as to be able to obtain automatic plots of enzyme activity against protein concentration. At the same time, we have combined the system with the technique of “multiple enzyme analysis” previously developed (2) so that groups of enzymes may be studied. We have also developed a FORTRAN computer program to calculate the instrument calibrations and the enzyme activities. METHODS

The techniques are illustrated by studies on ferricyanide reductases in yeast homogenates. Yeast strains. These were kindly supplied by Dr. D. Wilkie of the Department of Botany, University College London. Preparation of homogenates. Yeast was grown to stationary phase at 30” in yeast extract/peptone/2% glucose with vigorous shaking. The cells were washed three times with distilled water, and disrupted in 0.3 M mannitol, by shaking with glass beads in a Braun shaker (3). 496

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Protein nitrogen. Cells were treated according to the procedure of Schneider (4) and the final insoluble residue was assayed for total nitrogen by a micro-Kjeldahl method. Enzyme assays. A standard Technicon AutoAnalyzer was used, wit.h the arrangement, is shown in Figure 1. Substrate or substrates were placed on the sampler table (S). A sampler I module was used, and since there was no sample-wash facility, substrates were only placed in odd-numbered cups, and water was placed in all even-numbered cups. The substrate stream was mixed with buffer and cofactors and segmented with N, gas on the proportioning pump P. Stock enzyme was placed in the mixer Ml

FIG. 1. Manifold for multiple enzyme analysis with gradient-making system. C = continuous flow calorimeter, D=debubbler, HB= heating bath, Ml, M2= magnetic stirrers, MC = mixing coils, P = proportioning pump, S = sampler, and W = waste (see text for explanation).

in a bottle surrounded by crushed ice. The enzyme was pumped through line 6 into a suitable volume of diluent in a similar bottle on mixer M2, and the diluted “enzyme gradient?’ was mixed with substrate, buffer, and cofactors. The mixture was incubated in a heating bath HB and after debubbling (D) the optical density was read in a continuous-flow colorimeter C. The reaction mixture was then pumped to waste (W) by line 5. The gradient-making device (see below) consists of Ml, M2, and lines 4 and 6. Assays were carried out at 37”, and oxygen-free nitrogen was used as the gas phase. The standard buffer was 0.05M potassium phosphate, pH 7.4, and it, contained 0.8 mM K,Fe(CN), and 0.1% nonionic detergent (Triton x 100). The ferricyanide was used as an electron acceptor

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to follow the various dehydrogenases, and the enzyme reaction was followed by the fall in Ebzo. The detergent served to reduce turbidity of the reaction mixture and improve the bubble pattern. Details of the yeast homogenate employed, the line volumes and substrates used, and additions to the mixers Ml and M2 are given in the legends to the various figures. RESULTS

AND DISCUSSION

Gradient-making device. The gradient-making device is based on the system of Davis, Santen, and Agranoff (1). The shape of the gradient depends on the relative rates of emptying and filling of M2 (Fig. 2). The

Cup number

FIG. 2. Effect of relative rates of emptying and filling in shape of gradient. The manifold in Figure 1 was used with water in all lines. The flow rates (in ml/mm) were as follows: line 1, 0.6; line 2, 2.0; line 3, 1.2; line 4, 1.2; line 5, 2.5. Line 6 had the following flow rates: Expt. A, 2.0; Expt. B, 0.6; Expt. C, 0.16. Mixer 1 contained 2 mM K3Fe(CN)s and the gradients were started with 75 ml Hz0 in mixer 2. Sampling rate 40 cups/hr. gradient is linear when the rate of emptying of M2 is exactly twice the rate of filling. In this situation, the slope of the gradient is inversely proportional to the volume of diluent in M2 (Fig. 3). During the course of an experiment, the gradient-making system operates while samples are being aspirated from the sampler. It is therefore necessary to know how far the stock solution has been diluted at the

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moment that any given sample is being assayed. This is done as follows: A standard “marker” solution (e.g., 1 mM K,Fe(CN) ,) is placed in M2 and run directly into the AutoAnalyzer through line 4, with line 6 disconnected. Let us call the reading so obtained “stock reading.” M2 is then washed out and filled with a volume of water equal to that to be used by the diluent in the actual experiment; line 6 is connected and marker placed in Ml. Water is placed in all the cups, and the sampler and

F’IG. 3. Effect of volume of fluid in mixer 2 on slope of gradient. Conditiona aa in Figure 2 except that volume of water in mixer 2 varied from 20 to 160 ml. Flow rate of line 6 was 0.6 ml/min.

gradient-making system are started at the the sample mixes with the gradient of the density falls slightly, because of dilution reading for each cup can thus be identified. “gradient dilution factor (GDF) ” which is

same time. As the water in marker solution, the optical by the water. The gradient It is useful to use the term given by:

GDF = reading for a given cup stock reading A plot of gradient dilution factor against cup number is given in Figure 4. It can be seen that the values are reasonably linear. Automatic plot of enzym activity against protein concentration. If “stock” enzyme is placed in mixer 1 and a suitable diluent in mixer 2, a gradient in enzyme concentration is established. If substrates are placed in the sampler cups, one can obtain a direct plot of enzyme activity against protein concentration. Two examples will be given. In Figure 5, a plot of lactate dehydrogenase against cup number shows that only the

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first four values were linear. (Since the ‘(enzyme gradient” was linear, plotting activity against cup number is equivalent to plotting it against the actual protein concentration.) The experiment in Figure 5 is with a single enzyme, and, if substrate is fed in directly through line 1, without the use of the sampler, a continuous trace is attained. (A similar system has been described by Tappel and Beck (5) for p-glucuronidase.) 1.0 I

Cup number

FIQ. 4. Plot of gradient dilution factor (GDF) against cup number. Conditions as in Figure 2 with 50 ml water in mixer 2. Flow rate of line 6 was 0.6 ml/min. See text for method of calculation of GDF.

If repeating groups of substrates are placed on the sampler table, it is possible to obtain plots of activity against protein concentration for several enzymes, i.e., the system can be used as a “multienzyme analyzer.” An example of such a plot is given in Figure 6. Four enzyme are studied at the same time and it can be seen that only three of them gave linear results. NADH-ferricyanide reductase was not linear with respect to protein concentration for any of the assay points. The best estimate of enzyme activity in this case would therefore be given by the initial slope of the curve in Figure 6. The final protein concentration in Figure 6 is calculated as follows: The various lines in the manifold are calibrated by running marker K,Fe (CN) 8 through each one, with water in all the other lines. The value “fractional line volume (FLV)” is calculated from the expression: reading with marker

FLV = reading with marker The concentration to the initial

in line

in all lmes

of any component in the final reaction mixture is equal

concentration

in the stock

solution

multiplied

by the frac-

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tional line volume of the line used to pump the component. If the stock component is placed in the gradient-making system, however, the concentration in the reaction mixture at the nth cup is given by: stock concentration

X FLV of line from gradient maker X GDF,

The final protein concentration at any given cup is thus calculated from the protein concentration in mixer 1, the fractional line volume of line 4, and the gradient dilution factor for that cup, determined by previous calibration (Fig. 4). Since these calculations rapidly become tedious, particularly with nonlinear gradients, a computer program has been developed to calculate the calibrations and enzyme activities.

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Cup number

Fro. 6. Automatic test of linearity of lactate dehydrogenase away. Sampling and flow rates aa in Figure 4. Stock enzymes (0.51 mg protein N/ml homogenate in 0.3 M mannitol of yeast strain 46) in mixer 1; 50 ml 0.3 M mannitol in mixer 2; 0.05 M pottium phosphate, pH 7.4/0.3 mM KJ?e(CN),/O.l% Triton X-100 in line 2; oxygen-free nitrogen in line 3. The sampler table contained 0.01 M sodium L(+) -lactate in alternate cups.

Data processing and computer program. Our aim was to reproduce the flexibility of manual records as far as possible. Thus most of the program is concerned with the input and output of data in “open form,” i.e., using written text and chemical symbols, taking the order of substrates as they were sampled. Special numerical codes for the reagents and samples were avoided, and rigid tabulation of data was kept to a

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minimum. Internal checks and procedures for calibration and interpolation of data have been built into the program, thus minimizing the possibility of human error, but a large measure of human control has been retained over the design of the analyses. The program is written in FORTRAN, with input of data on punched cards, and output on a line-printer. An IBM 7090 computer was used. The program has about 450 statements and takes about one minute to compile and run. The following general description shows the scope of this particular combi-

Protein

concentration

(+g protem

N/ml)

FIQ. 6. Multiple enzyme analysis for four dehydrogenases. Conditions as in Figure 5. Stock enzyme (0.28 mg protein N/ml homogenate in 0.3 M mannitol of yeast strain D243) in mixer 1. Water in cups 1, 11, 21, and 31; 0.002 M NADH in cups 3, 13, 23, and 33; 0.01 M sodium L(+)-lactate in cups 5, 15, 25, and 35; 0.01 M sodium DC-)-lactate in cups 7, 17, 27, and 37; 0.01 M sodium succinate in cups 9, 19, 29, and 39. Ensyme activities and protein concentrations calculated by computer (see Table 1).

nation of AutoAnalyzer and computer. If the reader requires full details of the program, printout and card decks are available from the authors. The sequence of operations is as follows: 1. A description of the assay conditions and other relevant comments is presented first in free format. (This section is equivalent to the introductory notes in a laboratory notebook.) 2. The names and concentrations of the various reagents going into the various lines are read in, including the stock protein concentration of the enzyme mixture in mixer 2. 3. The concentration of the “marker” used in the gradient calibration

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is read in, followed by the reading and zeros with the marker, and then for each successive cup. The cups need not be in continuous sequence since the program will make linear interpolation for the missing cup numbers. 4. The names of the various substrates on the sampler table are read in, using free format. The names are read in any order, but each substrate is followed by its associated cup numbers and concentrations. The program checks that no cup number is outside the range of the gradient calibration in step 3 above. 5. The blank readings (i.e., the values with water instead of substrate in the sample cup) are read in, together with instrument zeros for each blank (i.e., the values with water in all lines, instead of reaction mixture). The program will interpolate blanks linearly for any missing cup numbers. 6. The actual readings of the enzyme assays for each substrate are then read in with the appropriate instrument zeros, 7. Data for calculation of fractional line volumes are read in next. The concentration of the marker solution, and the readings and zeros for each line are read in here together with the maker’s specification of the flow rate for each line. 8. The molar extinction coefficient is then calculated by pumping a series of standard solution of chromophore through one line, with water in all the other lines. The line number used and the initial concentrations of the chromophore are read in here, followed by the readings and zeros. 9. The time of incubation of enzyme with substrate is then read in. The results are then calculated as follows: (1) The zeros are subtracted from the readings for the data for calculation of molar extinction coefficient. These are tabulated against concentration and arranged for linear interpolation. (2) The gradient is next calibrated. Zeros are subtracted from the readings for the given cup numbers. These values are converted into molarities and the gradient dilution factor (GDP) is calculated for each cup, and an interpolated value calculated for missing cup numbers within the range of calibrations. (5) Readings for the calibration of the various lines are converted into molarities as above, and the fractional line volume (FLV) calculated for each line. This is checked against the FLV value calculated from the flow rates given by the manufacturer, and discrepancies greater than 10% are reported. (4) The readings are then converted into molarities for the given cups, after subtraction of the blanks.

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TABLE 1 Typical Computer Output RlXdta

Substrate

cup

Water

NADH

Na L(+)

Lactate

Na D(+)

Lactate

Na Succinate

no.

1 11 21 31 3 13 23 33 5 15 25 35 7 17 27 37 9 19 29 39

Substrate 00°C. (moles/ml)

Protein cone. (mg N/ml)

0

0.0008 0.0090 0.0173 0.0255 0.0025 0.0107 0.0190 0.0271 0.0041 0.0124 0.0206 0.0288 0.0057 0.0140 0.0222 0.0304 0.0074 0.0156 0.9238 0.0321

0.355

1.773

1.773

1.773

Enzyme

~molea/ml

0 0 0 0 0.0138 0.1273 0.1873 0.2242 0.0055 0.0185 0.0323 0.0369 0.0231 0.0646 0.0831 0.1163 0 0 0 0

activity

ptnolas min/m /

rmoles/min/ nrg pr N

0 0 0 0 0.0019 0.0175 0.0257 0.0308 0.0008 0.0025 0.0044 0.0051 0.0032 0.0089 0.0114 0.0160 0 0 0 0

0 0 0 0 0.7695 1.6381 1.3562 1.1351 0.1853 0.2052 0.2158 0.1762 0 5524 0.6343 0.5150 0.5246 0 0 0 0

1 Assay conditions Experiment 76 Determination of respiratory enzymes in yeast mutants. Investigation of ferricyanidereductases in petite mutants. Assay no. 76/5 Enzyme Yeast Genot,ype Gene petite Th 76/l D206,‘2B As3ay conditions Filter Wash cups Ma&oid Inc. temp. Coil no. Samples/hr E 37°C 1 40 420 rnp water fine no. Description 1 Sample 2 Buffer 3 Gas 4 Enzyme (from gradient maker) 5 Waste 6 (to gradient maker) units of 2 Reaction mixture Line Initial Final cont. no. cont. cont. 4 0.136 0.644 mg protein N/ml Enzyme (th 76/l) 2 0 . 800 0.399 ~moles/ml K Ferricyanide moles/ml K Phosphate pH 7.4 2 50.000 24.913 2 0.050 Per cent v/v Triton-X-100 0.100

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(6) After subtraction of the blanks for each of the cup numbers associated with each substrate in the sample pattern, the substrate and protein concentrations are calculated. For each cup the molar values of the readings are used to obtain the enzyme activity for the substrate in that cup in terms of pmoles/ml. This value is divided by the incubation time to give the activity in ,umoles/min/ml and by the protein concentration of that cup to give the activity in ~moles/min/mg protein N. A typical output table is given in Table 1. (The actual computer output was from a line-printer which only printed capital letters.) SUMMARY

1. By incorporating a gradient-making system into a Technicon AutoAnalyzer, an automated method has been developed for determining the relationship between enzyme activity and protein concentration. 2. This has been applied to a system of multiple enzyme analysis so that groups of enzymes of widely different activity can be assayed at the same time under conditions of linearity with respect to protein concentration. 3. The characteristics and method of calibration of the analytical system are described. A computer program is presented which calculates and tabulates the enzyme activities from data obtained during the calibration of the instrument, the gradient-making system and the pump tubing, as well as from the appropriate readings and blanks in the actual enzyme assay. ACKNOWLEDGMENTS The authors are grateful to Miss J. Jones and Mrs. D. Collier for technical assistance. They are also grateful to the .Medical Research Council and the Science Research Council for research grants and to Technicon Instrument Company, chertsey for the loan of some equipment. REFERENCES 1. DAMS, G. A., SANTEN, R. J., AND AGRANOFF,B. W., Anal. Biochem. 2. ROODYN, D. B., Nature 206, 1226 (1965). 3. ROODYN, D. B., AND WILRIE, D., Biochem. J. 103, 4. SCHNEIDER, W. C., J. Biol. Chem. 161, 293 (1945). 5. TAPPEL, A. L., AND BECK, C., in “Automation in

con Symposia 1965,” pp. 555-558. Mediad

11, 153 (1965).

3c (1967).

Analytical Chemistry: Inc., New York, 1965.

Techni-