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The automated assay of complete enzyme reaction rates
ANALYTICAL
22, 359-373
BIOCHEMISTRY
The
Automated
II. Digital HENRY
Readout
C. PITOT,’ McArdle
Laboratory,
(1968)
Assay Reaction and NANCY
of Complete Rates
Data
Processineg
WRATTEN,”
AND
The Medical School, University Madison, Wisconsin 69706 Received
December
Enzyme
of
linear
MIRIAM
Rates1 POIRIER”
of Wisconsin,
27. 1966
The first paper of this series (1) described a combination unit consisting of a Gilford Model 2000 Multiple Sample Absorbance Recorder and Technicon proportioning pump and large sampler. This unit was capable of assaying and recording the rates of a large number of enzymes completely automatically. Up to 300 assays could be performed in one 24 hr day and the results recorded as graphic rates on the recorder of the Model 2000. The disadvantage of the system was the rather laborious routine of plotting and calculating the rates from the graphic record. In most instances these calculations consumed 3- to 7-fold more working hours than did the assays themselves. In order to obviate this problem as well as improve the future utilization of the data, the combination unit has been equipped with a Non-Linear Systems Model 4206 digital voltmeter, system control and Model 180 output adaptor which feeds into an IBM 526 card punch. These additions permit the data to be coincidently punched on cards as it is being recorded, thus allowing for calculation of the data obtained by the use of computer techniques. This paper describes the electronic format and operation of the data processing system, as well as representative enzyme assays and the computer programs used in compilation and calculation of the punched data. METHODS
The general methodology for the use of the combination unit is as previously described (1). However certain modifications as well as the ‘This work was supported by grants from the National Cancer Institute (CA07175), the American Cancer Society (P-314 and In-35), and the University of Wisconsin Research Committee. ’ Career Development Awardee (CA-29, 405) of the National Cancer Institute. a Present address: University of Maine, Orono, Maine. 4 Present address: Institut du Cancer, HBpital Notre Dame, Montreal, P. Q,, Canada. 359 @ 1968 by
Academic
Press
Inc.
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addition of the data processing system have been made in the unit and will be described under instrumentation. Examples of assay procedures will be given in the following section. A. Instrumentation A photograph of the entire unit as it is now used is seen in Figure 1. The modifications from those previously described (1) are as follows: (a) the sample tubes are kept at 6-8°C or less in mater that circulates through a Haake thermoelectric cooler and insulated silicone tubing by means of a small pump (Eastern Industries, Model Al). The sample rack has been modified by rebuilding the upper layers of watertight soldered stainless steel to hold the circulating water. The Technicon proportioning
FIG. 1. Photograph of combination unit equipped for data processing of output. The system control Model 180 and the 4206 digital voltmeter are on above the Gilford 2000 chassis. The large cable to the IBM 526 punch extending from the left of the 180. The clock (A) controlling digitization rate as the scan delay (B) clock may be noted on the system control.
digital a shelf is seen as well
pump is the two-speed version and the fast speed is always used when the sample clock is running. This allows for a shortening of the sample time from the original 4 min (1) to only 1.5 min. An AutoAnalyzer manifold (Fig. 2) is employed to facilitate removal of the tubing should it be necessary to use the unit for purposes other than enzyme rate determination such as are mentioned in the discussion. In place of the glass mixing coils, jet mixers (Fig. 2) are employed for both mixing and cooling. These are made from standard AutoAnalyzer Tygon tubing. Pump tubing of
AtJTOiifATED
EN.k!&fE
BATES.
FROM
FIG.
enzyme Decimal
2. Format reaction numbers
361
Ii.
RESEVOlFi
of manifold and tubing used in automated rates. A closeup diagram of the jet mixer in parentheses refer to tubing diameter.
analysis of complete is seen in the insert.
0.035 in. diameter (Technicon Corporation) has been found adequate for the system described here. Type C debubblers (Technicon Corporation, Chauncey, New York) have been most useful in preventing air bubbles taken into t’he dip rods at the end of the sample time from entering the cells. However, for most efficient running of the system it is still necessary to deaerate the diluting and reservoir solutions prior to use. The jet mixers are immersed in ice as before, the ice container resting on a Plexiglas shelf extending from the undersurface of the pump and held attached by standard removable 9 in. legs. This permits easy access to the control knob and galvanometer of the Gilford absorbance meter. As seen in the picture (Fig. 1) Labline Duo clocks wit.h 12 hr timers are employed to automatically shut off the Therm0 cool and circulating pump, the large sampler, the Model 2000 and t.he Model 4206 Digital voltmeter when it is necessary to run the unit unattended. In Figure 1 may be seen the Non-Linear Systems data processing equipment including the System Control with its electronic clock which determines the digitizing rate (A) and the scan delay clock (B) which shuts off the punching during pump time and any lag time (vide infra) . The basic electronic circuitry for the attachment of the 4206 and
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system control to the Model 2000 is similar to that described by Krichevsky et al. (2). The four Potter Blum relays which give channel information are inside the System Control (Fig. 3). Of the display on the digital voltmeter only the digits are used to feed into the card punch. Thus each word consists of a letter, A, B, C, or D, and four numbers. The digitizing rate may be preset to take readings every 1, 2, 10, 20, or 60 set (other time intervals may be used by prior specification). This electronic digital clock in the System Control digitizes independently of the other functions of the unit except during the running of the scan delay clock or when either the dwell timer or auxiliary dwell timer on the Model 2000 are not running, i.e., during switching of the cuvet positioner from one sample to the next. The signal from the digital clock as well as that from the lights of the cuvet positioner which indicate the channels are sent to the digital voltmeter from the system control by means of a contract closure relay. The basic wiring component of the system control is seen in Figure 3. The wires carrying the signal coming from the large sampler are attached to the counter reset microswitch (see Technicon Automatic T/F Fraction Collector Telephone Manual). When the sampler rack is moved one step forward, a cam on the duocam trips the microswitch for a period of time sufficient to allow two or more (up to 4) impulses through to the card punch. When the relay (K5, Fig. 3) is tripped by this signal a -15 V signal is sent to the digital voltmeter which, having been set for a maximum of 10 V, sends a signal of 9999 to the 180 and this number is then punched. Occasionally at this point a double punch may occur or a voltage between 3.000 and 9.999 if the signal comes when the microswitch is only partially tripped. A double punch occurs so infrequently (less than once in 10,000 words except at high-frequency digitizing rates) as to be of negligible significance, and errors due to a low voltage signal are programmed out (vide infra). The purpose of the scan delay clock in the system control is to prevent readings being taken during the exchange of samples in the flowthrough cells, i.e., while the pump is running, and to allow for any lag in the initial rate of the reaction curve. The scan delay clock is set to the “on” position by the -15 V signal from the sampler. The connections placed in the optical density converter of the Model 2000 are identical to those of Krichevsky et al. (2). However it was necessary to make other adjustments to remove the ripple due to the AC voltage input. This was done by placing a voltage 180” out of phase across the input voltage from the 12AT7 tube (2). The ripple can be adjusted to a minimum by means of a 10 turn 100 K variable resistor set in the ripple adjust line. The voltage readings of the digital vohmeter
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.,..-..
FIG. 3. Diagram of major circuits in system control. of Non-Linear Systems, Inc., Del Mar, California.)
(Reproduced
by permission
are set to optical density readings equivalent with the absorbance meter using the optical density standards supplied by the Gilford Corporation and another 10 turn 100 K variable resistor set across the output lines of t,he V14 tube (2). To further decrease the ripple a 1 mpfarad condenser was placed across the input to the digital voltmeter. By means of these connections it is possible to reduce the ripple to about 0.001 to 0,002 optical density unit when the digital voltmeter is taking 50 readings per second at its maximal rate. During normal operation, timed readings are taken only when one of the dwell timers on the Model 2000 is running. This is
364
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WRATTEN,
AND
POIRIER
controlled by the connections of t,he clock motors to relays K13 and K15 (Fig. 3). A schematic diagram of the entire system is seen in Figure 4. In essence the operation of the basic unit is as described previously (1) in that four samples are pumped in simultaneously through precalibrated pump tubes. Each pair of pump tubes have equivalent flow rates to within 0.5% to ensure an equivalent delivery of enzyme and reactants. At the end, of the sample time the dip rods lift and, after the scan delay clock on the system control (B, Fig. 1) times out, optical density and channel information are punched at intervals preset by the digital clock. During
I
CUVETTE FOSITIONER
SYSTEM CONTROL AND TIMERS a
DIAGRAM OF COMBINATION UNm WITH DATA ACQUlSiTiON CGMPONENTS
FIG. 4. Over-all scheme of combination unit equipped with data processing of digital readout. The preamplifier (Preston Scientific, Model 8300) may be used to increase the sensitivity of the readings by a factor of 2, 5 or 10.
this period the cuvet positioner is switching positions at intervals preset by the dwell timer of the Model 2000. At times for slow reactions both dwell timers are used. As noted above, digital readings are taken only while the dwell timers are running. At the end of the total cycle time, the sample rack moves forward one position, rotating the cam which trips the microswitch in the sampler sending a 9999 from the digital voltmeter to the card punch. This signal also starts the scan delay clock, thus stopping further digital readings while the pump is running and bringing in new samples. The cycle is then repeated and so on. B. Assay Procedures The method of preparation of the tissues for enzyme assay is identical to that described previously (1). The dilution of the enzyme samples was performed with a mechanical autodilutor (Research Specialties Corp.),., The automatic dilutor was calibrated each day using a standard of 1 mM Y-AMP (Pabst Laboratories) prepared using an analytical balance. The method of calibration used in our laboratory is as follows: the volume
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of the diluting syringe of the automatic diluter was calibrated with a graduated cylinder or volumetric flask using a summation of multiple samplings. The volume of the sample syringe was then calibrated by dilution of the 5’-AMP solution and checked on a Beckman spectrophotometer against the optical density obtained with the same clilution of the standard solution performed volumetrically by hand. For calibration the optical denait#y of the hand diluted and machine diluted sample agreed within 0.59 of the total optical density reading of the diluted sample. To ensure the pumping of equal volumes of solutions from the sample t’ube and the reservoir, the pump tubing was calibrated using 5 ml graduates to collect the volume of liquid pumped in a 1.5 min period at the high speed of the pump. Two segments of tubing were paired when their pump volumes were equivalent to within less than 1% on 3 separate trials. For the most reproducible operation, pump tubing was changed every month. Tyrosine ar-ketoglutarate transaminase assay: The assay procedure used for this enzyme reaction was a modification of that described by Lin and Knox (3). The format of this and the other reaction assay described will be similar to that described in the init.ial publication (1). Reservoir: I M borate buffer, saturated (at 37’) with L-tyrosinc, pH 8.0. 2.5 X IO-” M diethyldithiocarbamate. 2.5 X lo-” M a-ketoglutarate (a stock solut.ion of 0.5 M a-ketoglutarate was prepared in water and the pH brought to 6.8 with sodium hydroxide; this stock solution was stored frozen and diluted l-20 in the assay buffer just before use). Individual sample tube: 0.05 M tris(hydroxymethyl)aminomcthane, lo-” M dithiothreitol, pH 8.0, with 0.025 unit of keto-enol tautomeraee per 3 ml. Each sample tube contains 3 ml with from 0.05 to 0.2 ml of an extract of a 20% homogenate of liver. The rates are determined at 310 mp. The clocks on the sampler are set for a 10 min total cycle and a 1.5 min pump time. The scan delay clock on the system control is set for 2.5 min and the digitizing rate is one reading per 2 sec. Samples with low to moderate activity may exhibit a slightly longer activation time requiring that the scan delay clock be set at 3 to 4 min. The rate is measured at 310 rnp. Glucose-6-phosphate dehydrogenase: 6-phosphogluconmate dehydrogenase: This m&hod is modified from that described by Glock and McLean (4). This method of assay takes into account the fact that the product of glucose-6-phosphate dehydrogenase is 6-phosphogluconate after the lactone ring is split. Thus the assay consists in running one
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WRATTEN,
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reaction at maximal velocity with both substrates and a second reaction rate with 6-phosphogluconate only as substrate. The difference in rates is the rate due to glucose 6-phosphate dehydrogenation. Reservoirs: 0.1 M tris containing 10e3 M EDTA, pH 8.0, with 10m4M dithiothreitol. 2.5 x 107’M KCI. 2.5 X 1O-3 M magnesium acetate. 2.5 X 1O-3 M nicotinamide. In addition to the above basic solution, the reservoir for the single assay tubes (6-phosphogluconic dehydrogenase alone) contains: 1.4 X 1O-3 M 6-phosphogluconate and 4.7 X 1O-4 M NADP. The reservoir for the double assay contains: 1.4 X 1O-3 M 6-phosphogluconate, 2.5 X 1O-3 M glucose 6-phosphate, and 9.4 X 10e4 M NADP. Highly active samples (those with activities of 500 pmoles per gram liver per hour or greater) tend to use up all available NADP and should be assayed using 6.5 X 10” M NADP in the single assay reservoir and 1.25 X 1O-3 M NADP in the double assay reservoir. Individual sample tubes: 0.1 M tris containing lob3 M EDTA and 10m4M dithiothreitol, pH 8.0. 6.25 x 1O-5 M NADP. In this assay the total volume in the tube is 2.0 ml, which contains 0.05 or 0.1 ml of an extract of a 20% homogenate of liver. Two individual sample tubes are pipetted and each pair is run with a different reservoir. The clocks on the sampler are set such that the total cycle time is 10 min and the sample time 1 min. The scan delay clock is set for 1.5 min with a digitizing rate of 2 sec. The rate is measured at 340 mp. Glucokinase-hexokinase: The basic assay procedure has been described (1). The clock settings are identical to those for the dehydrogenase assays described above. Reagents: L-Tyrosine, pyridoxal phosphate, a-ketoglutarate, and NADP were products of General Biochemieals Corporation. The sodium salt of diethyldithiocarbamate was obtained from Eastman Kodak. 6-Phosphogluconate as the barium salt, glucose-6-phosphate dehydrogenase, and dithiothreitol were obtained from California Biochemical Corporation. The barium was removed from the gluconate with KSO, before use. Glucose 6-phosphate as the sodium salt and keto-enol tautomerase were obtained from Sigma Corporation. RESULTS
A. Programs for Calculation of Digital Output As noted under ‘LMethods,” at each signal from the digital clock in the system control the optical density read at that time plus a code letter,
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A, B, C, or D, to indicate the channel, is punched on a card in the card punch. Thus the size of each word is 5 columns and each 80 column card contains 15 words, the initial columns being left blank for identification purposes. At the beginning of each series of assays on a single enzyme two control cards are hand punched, giving information which includes: the number of channels being read, the frequency of punching (1, 2, 10, or 20 set or 1 min), the direction of the rate reaction slope (positive or negative), the number of blanks, the enzyme type and the factor for converting from slope to pmoles/gm liver/hr. Enzyme type refers, basically, to the degree of complication of the enzyme assay: type 1 tells the computer to calculate the rates as they are; type 2 indicates that blanks run either at the beginning of t,hc assay or for each individual sample arc to be subtracted; type 3 instructs the computer to subtract channels 1 and 3 from 2 and 4, respectively, thus obtaining differential rates such as in the assay for GPD-PGD; type 4 is similar to type 3 except that a set of blanks run either at the beginning or simultaneously with each sample can be automatically subtracted from channels 1 and 3. In this latter manner are calculated the differential rates for the glucokinasehexokinase assay (1). The last half of the first card as well as the second card are used for descriptive purposes of the assay itself. The only other hand-punched information is the last, word on the final card, which must be hand-punched as a letter A, B, C, or D, and the numbers 9111. This clesignates the end of a series of assays on one enzyme and the computer now looks for a new pair of control cards ; if none is present, the calculation is finished. For the calculation of the linear reaction rates, two basic programs nrr used. Both are written in Fortran 63 for use in the Control Data Corporation 3600 computer.” The calculation of the actual rates or slope of the linear reaction is done by the method of least squares. The curve is fitted according to the following equations: ZY; = an + UlSX, ZXiYj = U&Xi + UIZSie which
may bc combined
and simplified
ZX
= Ul((ZXJ?
1-
5Copies
- z;xy
is the slope of the line, one obtains a _ ZXiI:Yi
The program
to give
(2X$
- z&&y; -
zx;
depicts the slope as a graphic function
of these
programs
are available
the relation-
on request
to the
on
an
senior
S and Y axis author.
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POIRIER
with the X axis being time and the Y axis optical density. In this way the various parameters may be summed and squared with the only limitation being that no more than 50 points may be fitted to an individual segment of the slope. The actual programs differ only in their method of calculation of the reaction rate. Program Q calculates reaction rates for each individual segment of the rate curve (Fig. 5A). On the
FIG. 5. Sample rates of tyrosine transaminase (A) and glucose-bphosphate dehydrogenase:6-phosphogluconate dehydrogenase (B). Four tissue levels are used in A and two levels, each with the double (‘) and single substrate level, in B. The heavy lined segments denote the portions of the curve used in the calculation of the computer output.
other hand, all of the rate segments are combined for slope fitting and calculation by Program R (Fig. 5B). Q calculates a value for each rate segment and this value is printed out; however, the maximum value is taken as the final value to be both printed and punched on cards. Calculations by this program are quite useful for enzymes which have a variable lag time before initiation of the rate or which rapidly exhaust the supply of substrate. A least mean square fit of all rate segments combined as calculated by Program R is best used with assays having a steady rate over a fairly long period of time. It is particularly useful in enzyme rates which are relatively low and for use in differential assays where one
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rate is subtracted from another (Fig. 5B). In all cases, a part of the output from the computer is the specific activity of the enzyme as calculated either by maximal rate calculations (Q) or by over-all curve fitting (R) . Punch card output may then be utilized in statistical or other programs for calculation of standard deviation and standard error or for plotting of t.he final data in specific formats. The plotted portion of the output is performed with a Calcomp plotter available in the University of Wisconsin Computing Center. All data storage is confined exclusively to printed and punched output. B. Sample Enzyme Assays In order to compare the precision and reproducibility of the manually calculated enzyme activities (1) with those calculated by the data processing apparatus, the enzymes tyrosine-rw ketoglutarate transaminasc (TAKG) , glucose-6-phosphate dehydrogenase : 6-phosphogluconate dehydrogenase (GPD-PGD) , and glucokinase-hexokinase (GK-HK) were utilized as being representative of the various types of rates and differential rate procedures calculated by the Programs Q and R. In all instances, as reported previously (I), blank rates before and after specific sample rates were not appreciably different, indicating negligible carryover of sample from one assay to the next. In Table 1 are seen the comparisons of the values obtained from four consecutive assays at different levels of liver high speed supernatant (S,). The values are given in specific activity, since that is the format of the output from the computer. It is obvious that, each va.luc is merely the product of the slope calculated from the least mean square equation and multiplied by a factor which takes into account the digitizing interval, t.hc extinction coefficient of the product, and the dilution factors involved. All enzyme samples were pipetted using an autodiluter. Pipetting of samples by hand gave essentially identical results. In the case of TAKG and GPD-PGD, blanks were run without substrate. For glucokinase-hexokinaee assay, the blank was as previously described (1). As can be seen from the data of Table 1, the values in general agree when the manual and computer calculated rat,es are compared. In some instances, the computer calculated rates are slightly higher than those found by manual calculation; however, this might be expected since the rates determined manually arc entirely dependent upon drawing the best line to fit, the graphic record. It c:m I)e notfed from the clat#n that tbc reaction rates for tyrosine transaminase are direct,ly proportional to the amount of liver S, assayed. However, in the case of the dual assays, the higher level of enzyme did not give quite twice the’ rate of the lower level. In the case of GPD-PGD,
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TABLE 1 Comparison of Manual and Computer Calculations: Enzyme Reaction Rates” Calculation
MaIld
1. Tyrosine Blank rate (before) Reaction rates 0.05 ml S8 0.10mlS3 0.15 ml S8 Blank rate (after) 2. Glucose-&phosphate Reaction rates 0.1 ml Sa (GPD) 0.1 ml Ss (PGD) 0.2 ml S8 (GPD) 0.2 ml S3 (PGD) Blank rate Hexokinase Glucokinase Blank rate Hexokinase Glucokinase
Computer
transaminase
1.2
2.1
63.2 _+ 1.6 131.1 * 1.5 186.4 f 3.9 1.7
66.6 5 0.6 130.9 * 1.0 192.4 f 2.1 2.4
dehydrogenase:6-phosphogluconate
49.7 43.9 96.7 70.9
f 1.3 f 1.3 + 1.2 rf: 1.2
3. Glucokinase-hexokinase (0.05 ml S,) 1.8 rate (0.05 ml S3) 4.9 + rate (0.05 ml SJ 79.7 f (0.1 ml S,) 5.4 rat.e (0.1 ml S3) 10.2 + rate (0.1 ml SB) 150.1 +
0.8 1.0 2.2 3.2
dehydrogenase
54.6 42.6 103.1 73.6
f f It +
1.7 3.5 + 84.1 + 5.2 6.6 + 156.6 +
2.7 0.5 1.8 2.0
1.8 4.5 0.6 8.6
6 All values are in pmoles product)/gm liver/hr + standard deviation of quadruplicate determinations. The second set of blank values for tyrosine transaminase were run after the reactions to demonstrate the lack of carryover in this procedure.
complete linearity with tissue may be obtained by increasing the substrates and cofactor by a factor of 5. However, the expense involved does not appear to warrant the slight increase in rate obtained. The values obtained under the conditions described herein are sufficient for most studies. In the case of glucokinase, enzyme saturation is never quite achieved, even at half-molar glucose levels. DISCUSSION
This system, which is an extension of the method described earlier (1)) affords the individual worker the means to carry out a large number and wide variety of enzyme assays with the output in a form easily amendable for further data processing. The format described may, of course, be modified by utilizing a somewhat different sampling device (Robert Zwick, personal communication) or different output modes such as paper tape, magnetic tape, or direct on-line connection with a computer. The
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present complete unit costs approximately $18,000. This does not include the card punch rental. Use of paper tape output and modified sampling devices may decrease the price to less than $15,000. The major advantage of the addition of a data acquisition system is the increase in output capacity, which had previously been limited by the manual calculations of multiple rates. Obviously, manual calculations are more subject to human error than the machine data, but as seen from Table 1 there is good agreement between the two methods when carefully done. A further advantage of the data processing unit described here is the output which allows for efficient storage of data, as well as its reul;ilization for a number of different types of calculations such as statistical, graphic, etc. In addition, kinetic measurements and curve fitting of enzyme rates that are not linear may be done with this unit and will be the subject of it future paper. Since the initial paper of this series (1)) a number of enzyme reaction rates have been determined with the combination unit. The rates which have been determined in this laboratory utilizing the systems described in the papers of this series are for serine dehydratase (L-serine hydrolyase, EC 4.2.1.13), tyrosine a-ketoglutarate transaminase (L-tyrosinc: 2-oxoglutarate aminotransferase, EC 2.6.1.5)) histidine a-ketoglutarate transaminase, phenylpyruvate tautomerase (phenylpyruvate keto-enolisomerase, EC 5.3.2.1)) tryptophan oxygenase (L-tryptophan : oxygen oxidoreductase, EC 1.13.1.12), formamidase (aryl-formylamine amidohydrolase, EC, 3.5.1.9)) histidase (L-histidine ammonia-lyase, EC 4.3.1.3)) urocanase, glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2)) phosphoribosyl pyrophosphate amidotransferase [ribosylamine-5-phosphate: pyrophosphate phosphoribosyltransferase (glutamate amidating) , EC 2.4.2.141, lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27), glucokinase (ATP: D-glucose 6-phosphotransferase, EC 2.7.1.2), hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1)) glucose6-phosphate dehydrogenase (n-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49), phosphogluconate dehydrogenase (6-phospho-n-gluconate: NAD (P) oxidoreductase, EC 1.1.1.43)) citrate-cleavage enzyme [ATP: citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephosphorylating) , EC 4.1.3.81, malate dehydrogenase (L-malate : NAD oxidoreductase, EC 1.1.1.37), “malic” enzyme (L-malate: NADP oxidoredurtase (decarboxylating) , EC 1 .l .1.40), fructokinase (ATP : n-fructose 6-phospho transferase, EC 2.7.1.4)) fructose aldolase (ketose-l-phosphate aldehyde-lyase, EC 4.1.2.7)) glycerokinase (ATP: glycerol phosphotransferase, EC 2.7.1.30), glutamate dehydrogenase (L-glutamate : NAD oxidoreductase (deaminating) , EC 1.4.1.2)) and alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC 1.1.1.1).
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This list may easily be expanded to include many other reaction rates, the only criterion being that a change in optical density proportional to the rate be recorded as a function of time. It is a relatively simple matter to do as few as four or eight, or as many as 300 or more, assays of a single enzyme. With fast reaction rates such as for glucose-6-phosphate dehydrogenase, it is possible to perform up to 48 assays per hour. For slower reaction rates the number performed per hour is obviously less. However, since the instrument requires a minimum of attention, rate measurements may be continued almost around the clock. Although the papers thus far have described primarily the use of the instrument in the monitoring of reaction rates, several other uses for the combination unit have now become apparent and are presently under study in this laboratory. By suitable manipulation of the times on the clocks, as well as the settings of the program card in the IBM 526 card punch, it is possible to record automatically on individual cards the optical density of individual solutions such as those from chemical determinations or other techniques in which a single value is necessary. In addition, the same unit may be utilized for monitoring chromat,ographic effluents, such as those obtained in amino acid chromatography or nucleotide analyses. By alternately monitoring the four channels, the chromatograms obtained may be analyzed by means of computer techniques. The calculations and techniques involved in these processes will be the subject of a future paper. SUMMARY
The components and operation of an automatic system for the assay and data compilation of enzyme reaction rates is described. Programs suitable for calculation of a large number of single and differential, as well as average and maximal, linear rates by the method of least squares are presented. Sample assays by manual and computer techniques are compared for the enzymes tyrosine cu-ketoglutarate transaminase, glucose6-phosphate dehydrogenase : 6-phosphogluconate dehydrogenase and glucokinase-hexokinase. Good agreement is obtained between the two methods. Possible other uses of the instrument are discussed. ACKNOWLEDGMENT The authors are deeply indebted to Messrs. Thomas Kurtzer and Jack Hale of Megatek, Inc., Chicago, Illinois, for their invaluable assistance in the establishment of the data processing components of the unit as a workable combination, and to Messrs. James Henkel and Michael Berman for writing the programs used with the combination unit.
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REFERENCES 1. 2. 3. 4.
PITOT, H. C., AND PRIES, N., Anal. Biochem. 9, 454 (1964). KRICHEVSKY, M. I., SCHWARTZ, J., AND MAGEE, M., Anal. B&hem. 12, 94 (1965). LIN, E. C. C., AND KNOX, W. E., Biochem. Biophys. Acta 26, 85 (1957). GLOCK, G. E., AND MCLEAN, P., Biochem. J. 5.5, 406 (1953).
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BIOCHEMISTRY
The
Automated
II. Digital HENRY
Readout
C. PITOT,’ McArdle
Laboratory,
(1968)
Assay Reaction and NANCY
of Complete Rates
Data
Processineg
WRATTEN,”
AND
The Medical School, University Madison, Wisconsin 69706 Received
December
Enzyme
of
linear
MIRIAM
Rates1 POIRIER”
of Wisconsin,
27. 1966
The first paper of this series (1) described a combination unit consisting of a Gilford Model 2000 Multiple Sample Absorbance Recorder and Technicon proportioning pump and large sampler. This unit was capable of assaying and recording the rates of a large number of enzymes completely automatically. Up to 300 assays could be performed in one 24 hr day and the results recorded as graphic rates on the recorder of the Model 2000. The disadvantage of the system was the rather laborious routine of plotting and calculating the rates from the graphic record. In most instances these calculations consumed 3- to 7-fold more working hours than did the assays themselves. In order to obviate this problem as well as improve the future utilization of the data, the combination unit has been equipped with a Non-Linear Systems Model 4206 digital voltmeter, system control and Model 180 output adaptor which feeds into an IBM 526 card punch. These additions permit the data to be coincidently punched on cards as it is being recorded, thus allowing for calculation of the data obtained by the use of computer techniques. This paper describes the electronic format and operation of the data processing system, as well as representative enzyme assays and the computer programs used in compilation and calculation of the punched data. METHODS
The general methodology for the use of the combination unit is as previously described (1). However certain modifications as well as the ‘This work was supported by grants from the National Cancer Institute (CA07175), the American Cancer Society (P-314 and In-35), and the University of Wisconsin Research Committee. ’ Career Development Awardee (CA-29, 405) of the National Cancer Institute. a Present address: University of Maine, Orono, Maine. 4 Present address: Institut du Cancer, HBpital Notre Dame, Montreal, P. Q,, Canada. 359 @ 1968 by
Academic
Press
Inc.
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addition of the data processing system have been made in the unit and will be described under instrumentation. Examples of assay procedures will be given in the following section. A. Instrumentation A photograph of the entire unit as it is now used is seen in Figure 1. The modifications from those previously described (1) are as follows: (a) the sample tubes are kept at 6-8°C or less in mater that circulates through a Haake thermoelectric cooler and insulated silicone tubing by means of a small pump (Eastern Industries, Model Al). The sample rack has been modified by rebuilding the upper layers of watertight soldered stainless steel to hold the circulating water. The Technicon proportioning
FIG. 1. Photograph of combination unit equipped for data processing of output. The system control Model 180 and the 4206 digital voltmeter are on above the Gilford 2000 chassis. The large cable to the IBM 526 punch extending from the left of the 180. The clock (A) controlling digitization rate as the scan delay (B) clock may be noted on the system control.
digital a shelf is seen as well
pump is the two-speed version and the fast speed is always used when the sample clock is running. This allows for a shortening of the sample time from the original 4 min (1) to only 1.5 min. An AutoAnalyzer manifold (Fig. 2) is employed to facilitate removal of the tubing should it be necessary to use the unit for purposes other than enzyme rate determination such as are mentioned in the discussion. In place of the glass mixing coils, jet mixers (Fig. 2) are employed for both mixing and cooling. These are made from standard AutoAnalyzer Tygon tubing. Pump tubing of
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EN.k!&fE
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FIG.
enzyme Decimal
2. Format reaction numbers
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RESEVOlFi
of manifold and tubing used in automated rates. A closeup diagram of the jet mixer in parentheses refer to tubing diameter.
analysis of complete is seen in the insert.
0.035 in. diameter (Technicon Corporation) has been found adequate for the system described here. Type C debubblers (Technicon Corporation, Chauncey, New York) have been most useful in preventing air bubbles taken into t’he dip rods at the end of the sample time from entering the cells. However, for most efficient running of the system it is still necessary to deaerate the diluting and reservoir solutions prior to use. The jet mixers are immersed in ice as before, the ice container resting on a Plexiglas shelf extending from the undersurface of the pump and held attached by standard removable 9 in. legs. This permits easy access to the control knob and galvanometer of the Gilford absorbance meter. As seen in the picture (Fig. 1) Labline Duo clocks wit.h 12 hr timers are employed to automatically shut off the Therm0 cool and circulating pump, the large sampler, the Model 2000 and t.he Model 4206 Digital voltmeter when it is necessary to run the unit unattended. In Figure 1 may be seen the Non-Linear Systems data processing equipment including the System Control with its electronic clock which determines the digitizing rate (A) and the scan delay clock (B) which shuts off the punching during pump time and any lag time (vide infra) . The basic electronic circuitry for the attachment of the 4206 and
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system control to the Model 2000 is similar to that described by Krichevsky et al. (2). The four Potter Blum relays which give channel information are inside the System Control (Fig. 3). Of the display on the digital voltmeter only the digits are used to feed into the card punch. Thus each word consists of a letter, A, B, C, or D, and four numbers. The digitizing rate may be preset to take readings every 1, 2, 10, 20, or 60 set (other time intervals may be used by prior specification). This electronic digital clock in the System Control digitizes independently of the other functions of the unit except during the running of the scan delay clock or when either the dwell timer or auxiliary dwell timer on the Model 2000 are not running, i.e., during switching of the cuvet positioner from one sample to the next. The signal from the digital clock as well as that from the lights of the cuvet positioner which indicate the channels are sent to the digital voltmeter from the system control by means of a contract closure relay. The basic wiring component of the system control is seen in Figure 3. The wires carrying the signal coming from the large sampler are attached to the counter reset microswitch (see Technicon Automatic T/F Fraction Collector Telephone Manual). When the sampler rack is moved one step forward, a cam on the duocam trips the microswitch for a period of time sufficient to allow two or more (up to 4) impulses through to the card punch. When the relay (K5, Fig. 3) is tripped by this signal a -15 V signal is sent to the digital voltmeter which, having been set for a maximum of 10 V, sends a signal of 9999 to the 180 and this number is then punched. Occasionally at this point a double punch may occur or a voltage between 3.000 and 9.999 if the signal comes when the microswitch is only partially tripped. A double punch occurs so infrequently (less than once in 10,000 words except at high-frequency digitizing rates) as to be of negligible significance, and errors due to a low voltage signal are programmed out (vide infra). The purpose of the scan delay clock in the system control is to prevent readings being taken during the exchange of samples in the flowthrough cells, i.e., while the pump is running, and to allow for any lag in the initial rate of the reaction curve. The scan delay clock is set to the “on” position by the -15 V signal from the sampler. The connections placed in the optical density converter of the Model 2000 are identical to those of Krichevsky et al. (2). However it was necessary to make other adjustments to remove the ripple due to the AC voltage input. This was done by placing a voltage 180” out of phase across the input voltage from the 12AT7 tube (2). The ripple can be adjusted to a minimum by means of a 10 turn 100 K variable resistor set in the ripple adjust line. The voltage readings of the digital vohmeter
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.,..-..
FIG. 3. Diagram of major circuits in system control. of Non-Linear Systems, Inc., Del Mar, California.)
(Reproduced
by permission
are set to optical density readings equivalent with the absorbance meter using the optical density standards supplied by the Gilford Corporation and another 10 turn 100 K variable resistor set across the output lines of t,he V14 tube (2). To further decrease the ripple a 1 mpfarad condenser was placed across the input to the digital voltmeter. By means of these connections it is possible to reduce the ripple to about 0.001 to 0,002 optical density unit when the digital voltmeter is taking 50 readings per second at its maximal rate. During normal operation, timed readings are taken only when one of the dwell timers on the Model 2000 is running. This is
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controlled by the connections of t,he clock motors to relays K13 and K15 (Fig. 3). A schematic diagram of the entire system is seen in Figure 4. In essence the operation of the basic unit is as described previously (1) in that four samples are pumped in simultaneously through precalibrated pump tubes. Each pair of pump tubes have equivalent flow rates to within 0.5% to ensure an equivalent delivery of enzyme and reactants. At the end, of the sample time the dip rods lift and, after the scan delay clock on the system control (B, Fig. 1) times out, optical density and channel information are punched at intervals preset by the digital clock. During
I
CUVETTE FOSITIONER
SYSTEM CONTROL AND TIMERS a
DIAGRAM OF COMBINATION UNm WITH DATA ACQUlSiTiON CGMPONENTS
FIG. 4. Over-all scheme of combination unit equipped with data processing of digital readout. The preamplifier (Preston Scientific, Model 8300) may be used to increase the sensitivity of the readings by a factor of 2, 5 or 10.
this period the cuvet positioner is switching positions at intervals preset by the dwell timer of the Model 2000. At times for slow reactions both dwell timers are used. As noted above, digital readings are taken only while the dwell timers are running. At the end of the total cycle time, the sample rack moves forward one position, rotating the cam which trips the microswitch in the sampler sending a 9999 from the digital voltmeter to the card punch. This signal also starts the scan delay clock, thus stopping further digital readings while the pump is running and bringing in new samples. The cycle is then repeated and so on. B. Assay Procedures The method of preparation of the tissues for enzyme assay is identical to that described previously (1). The dilution of the enzyme samples was performed with a mechanical autodilutor (Research Specialties Corp.),., The automatic dilutor was calibrated each day using a standard of 1 mM Y-AMP (Pabst Laboratories) prepared using an analytical balance. The method of calibration used in our laboratory is as follows: the volume
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of the diluting syringe of the automatic diluter was calibrated with a graduated cylinder or volumetric flask using a summation of multiple samplings. The volume of the sample syringe was then calibrated by dilution of the 5’-AMP solution and checked on a Beckman spectrophotometer against the optical density obtained with the same clilution of the standard solution performed volumetrically by hand. For calibration the optical denait#y of the hand diluted and machine diluted sample agreed within 0.59 of the total optical density reading of the diluted sample. To ensure the pumping of equal volumes of solutions from the sample t’ube and the reservoir, the pump tubing was calibrated using 5 ml graduates to collect the volume of liquid pumped in a 1.5 min period at the high speed of the pump. Two segments of tubing were paired when their pump volumes were equivalent to within less than 1% on 3 separate trials. For the most reproducible operation, pump tubing was changed every month. Tyrosine ar-ketoglutarate transaminase assay: The assay procedure used for this enzyme reaction was a modification of that described by Lin and Knox (3). The format of this and the other reaction assay described will be similar to that described in the init.ial publication (1). Reservoir: I M borate buffer, saturated (at 37’) with L-tyrosinc, pH 8.0. 2.5 X IO-” M diethyldithiocarbamate. 2.5 X lo-” M a-ketoglutarate (a stock solut.ion of 0.5 M a-ketoglutarate was prepared in water and the pH brought to 6.8 with sodium hydroxide; this stock solution was stored frozen and diluted l-20 in the assay buffer just before use). Individual sample tube: 0.05 M tris(hydroxymethyl)aminomcthane, lo-” M dithiothreitol, pH 8.0, with 0.025 unit of keto-enol tautomeraee per 3 ml. Each sample tube contains 3 ml with from 0.05 to 0.2 ml of an extract of a 20% homogenate of liver. The rates are determined at 310 mp. The clocks on the sampler are set for a 10 min total cycle and a 1.5 min pump time. The scan delay clock on the system control is set for 2.5 min and the digitizing rate is one reading per 2 sec. Samples with low to moderate activity may exhibit a slightly longer activation time requiring that the scan delay clock be set at 3 to 4 min. The rate is measured at 310 rnp. Glucose-6-phosphate dehydrogenase: 6-phosphogluconmate dehydrogenase: This m&hod is modified from that described by Glock and McLean (4). This method of assay takes into account the fact that the product of glucose-6-phosphate dehydrogenase is 6-phosphogluconate after the lactone ring is split. Thus the assay consists in running one
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reaction at maximal velocity with both substrates and a second reaction rate with 6-phosphogluconate only as substrate. The difference in rates is the rate due to glucose 6-phosphate dehydrogenation. Reservoirs: 0.1 M tris containing 10e3 M EDTA, pH 8.0, with 10m4M dithiothreitol. 2.5 x 107’M KCI. 2.5 X 1O-3 M magnesium acetate. 2.5 X 1O-3 M nicotinamide. In addition to the above basic solution, the reservoir for the single assay tubes (6-phosphogluconic dehydrogenase alone) contains: 1.4 X 1O-3 M 6-phosphogluconate and 4.7 X 1O-4 M NADP. The reservoir for the double assay contains: 1.4 X 1O-3 M 6-phosphogluconate, 2.5 X 1O-3 M glucose 6-phosphate, and 9.4 X 10e4 M NADP. Highly active samples (those with activities of 500 pmoles per gram liver per hour or greater) tend to use up all available NADP and should be assayed using 6.5 X 10” M NADP in the single assay reservoir and 1.25 X 1O-3 M NADP in the double assay reservoir. Individual sample tubes: 0.1 M tris containing lob3 M EDTA and 10m4M dithiothreitol, pH 8.0. 6.25 x 1O-5 M NADP. In this assay the total volume in the tube is 2.0 ml, which contains 0.05 or 0.1 ml of an extract of a 20% homogenate of liver. Two individual sample tubes are pipetted and each pair is run with a different reservoir. The clocks on the sampler are set such that the total cycle time is 10 min and the sample time 1 min. The scan delay clock is set for 1.5 min with a digitizing rate of 2 sec. The rate is measured at 340 mp. Glucokinase-hexokinase: The basic assay procedure has been described (1). The clock settings are identical to those for the dehydrogenase assays described above. Reagents: L-Tyrosine, pyridoxal phosphate, a-ketoglutarate, and NADP were products of General Biochemieals Corporation. The sodium salt of diethyldithiocarbamate was obtained from Eastman Kodak. 6-Phosphogluconate as the barium salt, glucose-6-phosphate dehydrogenase, and dithiothreitol were obtained from California Biochemical Corporation. The barium was removed from the gluconate with KSO, before use. Glucose 6-phosphate as the sodium salt and keto-enol tautomerase were obtained from Sigma Corporation. RESULTS
A. Programs for Calculation of Digital Output As noted under ‘LMethods,” at each signal from the digital clock in the system control the optical density read at that time plus a code letter,
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A, B, C, or D, to indicate the channel, is punched on a card in the card punch. Thus the size of each word is 5 columns and each 80 column card contains 15 words, the initial columns being left blank for identification purposes. At the beginning of each series of assays on a single enzyme two control cards are hand punched, giving information which includes: the number of channels being read, the frequency of punching (1, 2, 10, or 20 set or 1 min), the direction of the rate reaction slope (positive or negative), the number of blanks, the enzyme type and the factor for converting from slope to pmoles/gm liver/hr. Enzyme type refers, basically, to the degree of complication of the enzyme assay: type 1 tells the computer to calculate the rates as they are; type 2 indicates that blanks run either at the beginning of t,hc assay or for each individual sample arc to be subtracted; type 3 instructs the computer to subtract channels 1 and 3 from 2 and 4, respectively, thus obtaining differential rates such as in the assay for GPD-PGD; type 4 is similar to type 3 except that a set of blanks run either at the beginning or simultaneously with each sample can be automatically subtracted from channels 1 and 3. In this latter manner are calculated the differential rates for the glucokinasehexokinase assay (1). The last half of the first card as well as the second card are used for descriptive purposes of the assay itself. The only other hand-punched information is the last, word on the final card, which must be hand-punched as a letter A, B, C, or D, and the numbers 9111. This clesignates the end of a series of assays on one enzyme and the computer now looks for a new pair of control cards ; if none is present, the calculation is finished. For the calculation of the linear reaction rates, two basic programs nrr used. Both are written in Fortran 63 for use in the Control Data Corporation 3600 computer.” The calculation of the actual rates or slope of the linear reaction is done by the method of least squares. The curve is fitted according to the following equations: ZY; = an + UlSX, ZXiYj = U&Xi + UIZSie which
may bc combined
and simplified
ZX
= Ul((ZXJ?
1-
5Copies
- z;xy
is the slope of the line, one obtains a _ ZXiI:Yi
The program
to give
(2X$
- z&&y; -
zx;
depicts the slope as a graphic function
of these
programs
are available
the relation-
on request
to the
on
an
senior
S and Y axis author.
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with the X axis being time and the Y axis optical density. In this way the various parameters may be summed and squared with the only limitation being that no more than 50 points may be fitted to an individual segment of the slope. The actual programs differ only in their method of calculation of the reaction rate. Program Q calculates reaction rates for each individual segment of the rate curve (Fig. 5A). On the
FIG. 5. Sample rates of tyrosine transaminase (A) and glucose-bphosphate dehydrogenase:6-phosphogluconate dehydrogenase (B). Four tissue levels are used in A and two levels, each with the double (‘) and single substrate level, in B. The heavy lined segments denote the portions of the curve used in the calculation of the computer output.
other hand, all of the rate segments are combined for slope fitting and calculation by Program R (Fig. 5B). Q calculates a value for each rate segment and this value is printed out; however, the maximum value is taken as the final value to be both printed and punched on cards. Calculations by this program are quite useful for enzymes which have a variable lag time before initiation of the rate or which rapidly exhaust the supply of substrate. A least mean square fit of all rate segments combined as calculated by Program R is best used with assays having a steady rate over a fairly long period of time. It is particularly useful in enzyme rates which are relatively low and for use in differential assays where one
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rate is subtracted from another (Fig. 5B). In all cases, a part of the output from the computer is the specific activity of the enzyme as calculated either by maximal rate calculations (Q) or by over-all curve fitting (R) . Punch card output may then be utilized in statistical or other programs for calculation of standard deviation and standard error or for plotting of t.he final data in specific formats. The plotted portion of the output is performed with a Calcomp plotter available in the University of Wisconsin Computing Center. All data storage is confined exclusively to printed and punched output. B. Sample Enzyme Assays In order to compare the precision and reproducibility of the manually calculated enzyme activities (1) with those calculated by the data processing apparatus, the enzymes tyrosine-rw ketoglutarate transaminasc (TAKG) , glucose-6-phosphate dehydrogenase : 6-phosphogluconate dehydrogenase (GPD-PGD) , and glucokinase-hexokinase (GK-HK) were utilized as being representative of the various types of rates and differential rate procedures calculated by the Programs Q and R. In all instances, as reported previously (I), blank rates before and after specific sample rates were not appreciably different, indicating negligible carryover of sample from one assay to the next. In Table 1 are seen the comparisons of the values obtained from four consecutive assays at different levels of liver high speed supernatant (S,). The values are given in specific activity, since that is the format of the output from the computer. It is obvious that, each va.luc is merely the product of the slope calculated from the least mean square equation and multiplied by a factor which takes into account the digitizing interval, t.hc extinction coefficient of the product, and the dilution factors involved. All enzyme samples were pipetted using an autodiluter. Pipetting of samples by hand gave essentially identical results. In the case of TAKG and GPD-PGD, blanks were run without substrate. For glucokinase-hexokinaee assay, the blank was as previously described (1). As can be seen from the data of Table 1, the values in general agree when the manual and computer calculated rat,es are compared. In some instances, the computer calculated rates are slightly higher than those found by manual calculation; however, this might be expected since the rates determined manually arc entirely dependent upon drawing the best line to fit, the graphic record. It c:m I)e notfed from the clat#n that tbc reaction rates for tyrosine transaminase are direct,ly proportional to the amount of liver S, assayed. However, in the case of the dual assays, the higher level of enzyme did not give quite twice the’ rate of the lower level. In the case of GPD-PGD,
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TABLE 1 Comparison of Manual and Computer Calculations: Enzyme Reaction Rates” Calculation
MaIld
1. Tyrosine Blank rate (before) Reaction rates 0.05 ml S8 0.10mlS3 0.15 ml S8 Blank rate (after) 2. Glucose-&phosphate Reaction rates 0.1 ml Sa (GPD) 0.1 ml Ss (PGD) 0.2 ml S8 (GPD) 0.2 ml S3 (PGD) Blank rate Hexokinase Glucokinase Blank rate Hexokinase Glucokinase
Computer
transaminase
1.2
2.1
63.2 _+ 1.6 131.1 * 1.5 186.4 f 3.9 1.7
66.6 5 0.6 130.9 * 1.0 192.4 f 2.1 2.4
dehydrogenase:6-phosphogluconate
49.7 43.9 96.7 70.9
f 1.3 f 1.3 + 1.2 rf: 1.2
3. Glucokinase-hexokinase (0.05 ml S,) 1.8 rate (0.05 ml S3) 4.9 + rate (0.05 ml SJ 79.7 f (0.1 ml S,) 5.4 rat.e (0.1 ml S3) 10.2 + rate (0.1 ml SB) 150.1 +
0.8 1.0 2.2 3.2
dehydrogenase
54.6 42.6 103.1 73.6
f f It +
1.7 3.5 + 84.1 + 5.2 6.6 + 156.6 +
2.7 0.5 1.8 2.0
1.8 4.5 0.6 8.6
6 All values are in pmoles product)/gm liver/hr + standard deviation of quadruplicate determinations. The second set of blank values for tyrosine transaminase were run after the reactions to demonstrate the lack of carryover in this procedure.
complete linearity with tissue may be obtained by increasing the substrates and cofactor by a factor of 5. However, the expense involved does not appear to warrant the slight increase in rate obtained. The values obtained under the conditions described herein are sufficient for most studies. In the case of glucokinase, enzyme saturation is never quite achieved, even at half-molar glucose levels. DISCUSSION
This system, which is an extension of the method described earlier (1)) affords the individual worker the means to carry out a large number and wide variety of enzyme assays with the output in a form easily amendable for further data processing. The format described may, of course, be modified by utilizing a somewhat different sampling device (Robert Zwick, personal communication) or different output modes such as paper tape, magnetic tape, or direct on-line connection with a computer. The
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present complete unit costs approximately $18,000. This does not include the card punch rental. Use of paper tape output and modified sampling devices may decrease the price to less than $15,000. The major advantage of the addition of a data acquisition system is the increase in output capacity, which had previously been limited by the manual calculations of multiple rates. Obviously, manual calculations are more subject to human error than the machine data, but as seen from Table 1 there is good agreement between the two methods when carefully done. A further advantage of the data processing unit described here is the output which allows for efficient storage of data, as well as its reul;ilization for a number of different types of calculations such as statistical, graphic, etc. In addition, kinetic measurements and curve fitting of enzyme rates that are not linear may be done with this unit and will be the subject of it future paper. Since the initial paper of this series (1)) a number of enzyme reaction rates have been determined with the combination unit. The rates which have been determined in this laboratory utilizing the systems described in the papers of this series are for serine dehydratase (L-serine hydrolyase, EC 4.2.1.13), tyrosine a-ketoglutarate transaminase (L-tyrosinc: 2-oxoglutarate aminotransferase, EC 2.6.1.5)) histidine a-ketoglutarate transaminase, phenylpyruvate tautomerase (phenylpyruvate keto-enolisomerase, EC 5.3.2.1)) tryptophan oxygenase (L-tryptophan : oxygen oxidoreductase, EC 1.13.1.12), formamidase (aryl-formylamine amidohydrolase, EC, 3.5.1.9)) histidase (L-histidine ammonia-lyase, EC 4.3.1.3)) urocanase, glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2)) phosphoribosyl pyrophosphate amidotransferase [ribosylamine-5-phosphate: pyrophosphate phosphoribosyltransferase (glutamate amidating) , EC 2.4.2.141, lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27), glucokinase (ATP: D-glucose 6-phosphotransferase, EC 2.7.1.2), hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1)) glucose6-phosphate dehydrogenase (n-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49), phosphogluconate dehydrogenase (6-phospho-n-gluconate: NAD (P) oxidoreductase, EC 1.1.1.43)) citrate-cleavage enzyme [ATP: citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephosphorylating) , EC 4.1.3.81, malate dehydrogenase (L-malate : NAD oxidoreductase, EC 1.1.1.37), “malic” enzyme (L-malate: NADP oxidoredurtase (decarboxylating) , EC 1 .l .1.40), fructokinase (ATP : n-fructose 6-phospho transferase, EC 2.7.1.4)) fructose aldolase (ketose-l-phosphate aldehyde-lyase, EC 4.1.2.7)) glycerokinase (ATP: glycerol phosphotransferase, EC 2.7.1.30), glutamate dehydrogenase (L-glutamate : NAD oxidoreductase (deaminating) , EC 1.4.1.2)) and alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC 1.1.1.1).
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This list may easily be expanded to include many other reaction rates, the only criterion being that a change in optical density proportional to the rate be recorded as a function of time. It is a relatively simple matter to do as few as four or eight, or as many as 300 or more, assays of a single enzyme. With fast reaction rates such as for glucose-6-phosphate dehydrogenase, it is possible to perform up to 48 assays per hour. For slower reaction rates the number performed per hour is obviously less. However, since the instrument requires a minimum of attention, rate measurements may be continued almost around the clock. Although the papers thus far have described primarily the use of the instrument in the monitoring of reaction rates, several other uses for the combination unit have now become apparent and are presently under study in this laboratory. By suitable manipulation of the times on the clocks, as well as the settings of the program card in the IBM 526 card punch, it is possible to record automatically on individual cards the optical density of individual solutions such as those from chemical determinations or other techniques in which a single value is necessary. In addition, the same unit may be utilized for monitoring chromat,ographic effluents, such as those obtained in amino acid chromatography or nucleotide analyses. By alternately monitoring the four channels, the chromatograms obtained may be analyzed by means of computer techniques. The calculations and techniques involved in these processes will be the subject of a future paper. SUMMARY
The components and operation of an automatic system for the assay and data compilation of enzyme reaction rates is described. Programs suitable for calculation of a large number of single and differential, as well as average and maximal, linear rates by the method of least squares are presented. Sample assays by manual and computer techniques are compared for the enzymes tyrosine cu-ketoglutarate transaminase, glucose6-phosphate dehydrogenase : 6-phosphogluconate dehydrogenase and glucokinase-hexokinase. Good agreement is obtained between the two methods. Possible other uses of the instrument are discussed. ACKNOWLEDGMENT The authors are deeply indebted to Messrs. Thomas Kurtzer and Jack Hale of Megatek, Inc., Chicago, Illinois, for their invaluable assistance in the establishment of the data processing components of the unit as a workable combination, and to Messrs. James Henkel and Michael Berman for writing the programs used with the combination unit.
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REFERENCES 1. 2. 3. 4.
PITOT, H. C., AND PRIES, N., Anal. Biochem. 9, 454 (1964). KRICHEVSKY, M. I., SCHWARTZ, J., AND MAGEE, M., Anal. B&hem. 12, 94 (1965). LIN, E. C. C., AND KNOX, W. E., Biochem. Biophys. Acta 26, 85 (1957). GLOCK, G. E., AND MCLEAN, P., Biochem. J. 5.5, 406 (1953).
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