A simple device for automated spectrophotometric kinetics using a diode array spectrophotometer

ANALYTICAL

BIOCHEMISTRY

165,258-268

(1987)

A Simple Device for Automated Spectrophotometric Using a Diode Array Spectrophotometer ROBERTKSCOPES'ANDBARTON

Kinetics

HOLMQUIST’

Department of Pharmacology and Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115 Received April 23, 1987 In this report we describe an automated system that rapidly and automatically mixes reagents and records results, such as spectrophotometric changes. It employs a commercial diode array spectrophotometer and a novel dilution chamber in a flow stream that allows repetitive spectrophotometric rate measurements at accurately measured incremental substrate concentrations. When applied to enzyme kinetic studies, initial velocities at 15 different substrate or inhibitor concentrations, or pH values, can be recorded in a few minutes with high reproducibility, i.e., standard deviations less than l%, and high sensitivity. Reactions occur in an S-4 flow cell and the reagent consumption is minimal. The concentration of incrementally diluted reagent in the cell is measured directly by means of an indicator dye added to the substrate. Michaelis-Menten parameters, inhibition constants, and pH profiles are determined for several enzymes including dehydrogenases producing NADH, a kinase requiring a coupled assay, and a hydrolase, carboxypeptidase A, in a reaction that produces a small decrease in absorbance. 0 1987 Academic Press, Inc. KEY

WORDS:

enzyme kinetics; automation; diode array.

Many biochemical analyses, especially in enzymology, require systematic variation of the concentration of one component to determine its effect on a reaction. Common examples include the variation of substrates and inhibitors to establish their kinetic parameters and modes of inhibition and the variation of pH. Such measurements are usually performed manually, for example, by preparing separate reagent dilutions in cuvettes each containing a different substrate concentration and performing a series of individual reactions. This method is time consuming and can be subject to many errors. An automated system that would not only both mix reagents and record rates rapidly, as in the case of stopped flow, but also repeat the process automatically with different, accurately measured substrate concentrations, ’ Permanent address: Department of Biochemistry, Iatrobe University, Bundoora, Victoria 3083, Australia. 2 To whom correspondence should be addressed. 0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. All tights of reproduction in any form resewed.

could greatly facilitate data acquisition while improving precision. We here describe dilution analysis through a novel principle that employs a simple mixing block to achieve reagent dilution. It is implemented in conjunction with a diode array spectrophotometer that enables rapid measurement of reaction rates at one or more wavelengths and simultaneously measures substrate concentration at another. The instrument incrementally dilutes the reagent, mixes it with another component, e.g., enzyme or nucleophile, and initiates measurement within a few seconds of mixing. This is successively repeated over a range of concentrations suitable for calculating enzymatic parameters such as K,,, and k,,, inhibition and binding constants, and pH profiles. By adding an indicator dye to the substrate solution as an internal standard, it is possible to simultaneously measure both the substrate concentration in the reaction mixture and the reaction rate. 258

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METHODS

Chemicals. Alcohol dehydrogenase (ADH)3 isozymes from human liver were prepared and assayed as reported (1,2). Isolation of pyruvate kinase and preparation of ADH-1 and -2 from Zymomonas mobilis were performed as described (3,4); lactate dehydrogenase (pig heart) and carboxypeptidase A were obtained from Sigma, as were substrates and coenzymes for the enzymes. Rhodamine B and bromthymol blue were obtained from Aldrich, A Gilford 250 and a Hewlett-Packard 845 1A spectrophotometer were used for spectral measurements. One unit of enzyme activity is defined as the amount of enzyme required to transform 1 pmol of substrate per minute. Substrates for carboxypeptidase- A were obtained from earlier studies (5,6). Apparatus principle and design. The basic principle of the apparatus is diagrammed in Fig. 1 and a photograph of the device is shown in Fig. 2. It includes two glass syringes (E and S) driven by a computer-controlled linear drive. One syringe (S), containing the component to be varied, connects to double mixing chambers (B), approx 1 ml volume each, containing small magnetic stirring bars. The chambers are initially filled with buffer or water and sealed without any air spaces. The outlet from the second of these chambers connects to the output from the other syringe (E), containing, for example, enzyme, and the two flows meet. The liquids mix by passage along narrow channels (M), splitting and recombining several times to achieve homogeneity, before passing to an 89~1 flow cell positioned in the beam of the diode array spectrophotometer (HewlettPackard 8451A). A pulse of approximately 100 ~1 from each syringe is driven through the apparatus in about 1.5 s. With consecutive pulses, concentrated 3 Abbreviations used: ADH, alcohol dehydrogenase; Fa, 2-furanacryloyl; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid; Tricine, tris(hydroxymethyl)methylglycine; Mops, 3-morpholinepropanesulfonic acid.

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KINETICS

259

substrate from the drive syringe (S) enters the first mixing chamber, while less concentrated substrate simultaneously enters the second chamber. The solutions enter and exit the chambers at opposite ends, top and bottom, so that during the pulse only the mixed solution from the previous pulse exits; the new undiluted substrate entering the chamber is mixed during the rate measurement. The mixed enzyme and substrate pass into the flow cell. The substrate concentration in the flow cell is measured by comparison to an indicator dye, which should not inhibit or otherwise affect the enzyme being measured. Rhodamine B is highly suited for reactions that follow NAD/NADH redox reactions because of its high molar absorptivity (ca. lo5 M-’ cm-‘) and sharp peak (X,, = 552 nm), with virtually no absorbance at 340 nm where NADH measurements are made. Dye at an absorbance of 1.O (10 PM) at 552 nm is added to the substrate in the syringe. Readings near 340,440, and 552 nm are recorded every 0.5 s; the rate is calculated from measurements of A340-A440, and the substrate concentration is calculated from the mean of the measurements of A552-A440.Errors due to fluctuations in the output of the diodes are minimized by averaging absorption measurements at several wavelengths around the absorption maxima; baseline drift is largely eliminated by subtracting similarly averaged measurements near 440 nm. The time range for the rate is chosen according to the amount of enzyme used and the sensitivity of measurement; also any lags, such as occur in coupled assays, must be allowed for. For simple dehydrogenases, we have averaged the rates measured between 2 and 10 s, up to

FIG. I. Schematic diagram of the dilution showing the basic principle of its operation.

analyzer

FIG. 2. (A) Photograph of the dilution analyzer in position adjacent to the Hewlett-Packard 8451A showing the basic components of the entire system, including the linear transport, drive syringes, reservoir syringes, the block containing the mixing chambers, magnetic stirrer, and the 5ow cell in the sample compartment of the spectrophotometer. (B) Closeup of the block showing the chambers, mixing channels, and components to seal the chambers (delrin plugs, O-rings, and locking arm).

260

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linear increments of added compound, more suited for titration studies or pH variation (see below). There are many ways of organizing the syringe drives, which are controlled by the spectrophotometer. We have used 2 X l-ml syringes pushed by the linear cell transport drive of the Hewlett-Packard instrument. For most studies, 10 pulses of equal volume, followed by steadily increasing pulse volumes are used. To achieve highest substrate variation the syringes are refilled for a second set of pulses, making 2 X 2 ml the total volume. The final concentration of substrate emerging from the second mixing chamber after 2 ml is close to 60% that of the concentration in the syringe; thus the highest concentration in the assay is up to 30% that of c = Co[ 1 - (1 + vJvc)eeVdKJ, [II the concentration in the syringe. For simple where Co = concentration of substrate in the enzyme kinetics, the ideal substrate concensyringe, V, = volume of each mixing tration in the syringe is 10 to 15 times the chamber, and V, = total volume of pulses expected K,,, value. passed through. We have also used a single Programming of the drive system and kichamber apparatus, which gives closer to netics software. The software was developed for enzyme kinetics and can be easily modified for alternative procedures, such as inhibitor titration or pH variation (see below). The flow diagram of the program is shown in Fig. 4. The STEP# instruction, which controls the linear cell transport of the spectrophotometer, provides approximately 4500 individual steps for the full l-ml syringe plunge. Short pulses of not more than 80 steps were used to prevent back-pressure buildup, which strained the cell transport drive. Most of the experiments shown here used 5 pulses of 65 steps for each substrate increment, delivering 85 ~1 from each syringe; this took approximately 1.5 s. After FIG. 3. Values of substrate concentration (determined the syringes are refilled for the second set of from the indicator dye absorption) plotted against the pulses, the program directs more pulses to total volume of liquid passed through the mixing chambers. Top line, for single mixing chamber of 2.3-ml give larger substrate increments. Absorption volume. Lower line, for two 1.25-m] mixing chambers. measurements are integrated over 0.3 s and The lines are theoretical and calculated according to Eq. repeated every 0.5 s, and data at the appro[I] (lower line) or C = Ca( 1 - e-v”i’c) (upper line). The priate wavelengths are selected for calculascale of substrate concentration represents the fraction of the syringe concentration as it emerges from the mix- tion (see above). Rates are calculated based on linear AA vs time plots using all points ing chamber, which is further diluted twofold on mixing with enzyme. after 2 s. those between 2 and 50 s. Data collection begins 2 s after the pulse to ensure a static solution in the flow cell. Very satisfactory results have been obtained by taking measurements every 0.5 s for 10 s with the largest AA as small as 0.02. A 15-point kinetic run with 15 different substrate concentrations can be completed in a few minutes. The use of two chambers in series allows a spread of substrate concentration which starts low but increases exponentially, and later more linearly (Fig. 3). This arrangement is optimal for stepwise substrate concentration increments for enzyme kinetic studies. Theoretically, the concentration of substrate (c) emerging from the second chamber can be shown by simple calculus to be

262

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Calculate

Plot

Eadlr-Hof.trr

rms,

Plot

Km,

” “a

and

.

Vmax

Plot

Linew.sver-Blwk

FIG. 4. Flow diagram of the computer program, written in BASIC, used to control the dilution analyzer.

The program allows for rejection of obvious bad points on an initial Eadie-Hofstee display. Subsequently, the best-fitting curve to the v versus s plot is calculated by an iterative root mean square process (7). Having found K, and V,, , usually reliable to + 10%) plots are displayed as v versus S, Lineweaver-Burk, or Eadie-Hofstee (see Fig. 6). Construction. The mixing chamber block (Fig. 2), which is composed of one or two mixing chambers (Fig. 2B), and the flow mixer are made of plexiglass. The chambers are drilled (1.1 cm i.d.) and counter bored (1.27 cm i.d.) to provide a lip to hold the 1.27-cm O-ring seal. The plugs, cut from 1.2-cm-diameter delrin rod, are clamped tightly by pressure from above by the swing arm to seal the chambers. The flow mixer is constructed by milling channels approximately 1 mm deep by 1 mm wide in a scrap of .3/ 16-in. plexiglass and then welding it to the block with ethylene dichloride. Drilled channels, 1.6 mm in diameter, direct the liq-

uid flow between the various sections of the block. Plastic Luer fittings obtained from disposable needles are epoxied to the syringe mount holes to accept disposable three-way valves (Medex Inc., Hilliard, OH). The valves are oriented such that the reservoir syringes are upright. An air-driven magnetic stirrer (The Chemical Rubber Co., Cleveland, OH) is placed under the block to drive the stirring bars in both chambers simultaneously. The syringe drive mechanism used is the digital stepping motor linear cell transport of the Hewlett-Packard 845 1A spectrophotometer, mounted on a block in the appropriate position to drive both syringes for their entire length. The solution exiting the mixer passes through 0.5-mm-i.d. steel tubing to a l-cm path length Altex analytical optical unit flow cell that is mounted on a standard Hewlett-Packard 845 IA adjustable cuvette holder and aligned in the beam of the instrument. For NADH measurements, a uv filter of Pyrex glass is placed over the en-

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263

KINETICS

trance to the cell to prevent photodegradation of the coenzyme. RESULTS

General Procedure Using the instrument setup described under Materials and Methods, we have investigated its versatility by applying it to several enzymatic determinations. To operate, a solution containing the enzyme in buffer and any other components necessary for the reaction, except the substrate to be varied, is placed in the enzyme reservoir syringe. The component concentrations are made up to be twice the desired final values in the actual assay mixture delivered to the flow cell. The solution containing the substrate to be varied, and containing rhodamine B or other dye at an accurately known absorbance ( 1.O), is placed in the substrate syringe. For some procedures, a third solution goes into the mixing chambers-for instance, the substrate if an inhibitor is the variable. In most cases, however, water or a buffer of the same composition as the substrate solvent is used. After filling the drive syringes from the reservoir syringes and partially discharging them through the block to flush the channels, the mixing chambers are cleaned out, rinsed, filled with the required solution, and sealed. The valves are then directed to open the drive syringes to the system and the run is commenced.

Determination of K,,, and V,,,, Values of ADH A mixture containing buffer and NAD’ (each twice the normal concentration) and enzyme (typically 0.0 1 to 0.2 U/ml) was used in the enzyme syringe, and ethanol was used as substrate. Results obtained using a total of only 20 mU of enzyme are shown in Fig. 5, which also includes points obtained manually using the same solutions. The agreement of the two methods is excellent. A set of results using more enzyme, with consequently larger AA values, is shown in Fig. 6,

I

2

I/S

FIG. 5. Points obtained on the automated system (0) compared with those from the manual determination (0): human ADH isozyme ~$3,; ethanol, 20 mM; and NAD+, 2.4 mM, as substrates at pH 10 in 0.1 M glycine. This experiment was near the limit of sensitivity, with a AA3, after 50 s of less than 0.01 in each assay. The concurrence of the automated and manual points shows that rhodamine B does not affect the assay. Units of v and s are U/ml and mM, respectively.

in which the output of the spectrophotometer display is reproduced. Figure 6A shows the “fan” of rates successively obtained as the substrate concentrations incremented 17 times. Figures 6B, 6C, and 6D show these rates plotted in v against S, LineweaverBurk, and Eadie-Hofstee forms. Kinetic parameters were also measured for the reduction reaction using benzaldehyde as substrate. Enzyme, ?r-ADH, in 100 IIIM phosphate, pH 7.4, containing 100 PM NADH, and an aqueous solution of benzaldehyde were used in the enzyme and substrate syringes, respectively. Thus, an initial absorbance due to NADH of 0.31 was present in the cell and a decrease in absorption was monitored. The results (not shown) were comparable in quality and precision to those observed in the oxidation direction.

Reproducibility of Rate and Concentration A4easurernent.s The precision in reproducing the quantities delivered from each syringe, and the performance of the spectrophotometer, were tested by placing substrate in the chambers in addition to the substrate drive syringe for a complete experiment. With ADH in the en-

264

SCOPES AND HOLMQUIST

IO

20 TIME,

s

PIG. 6. Computer printouts for the reaction of fl,fi, ADH with 18 different ethanol concentrations at pH 10. (A) Progress curves. The first three rates were ignored in calculations as the system requires at least two pulses to flush it reliably. The display graphics automatically sets the first point to zero A. (B) Display of the v vs s plot. (C) Lineweaver-Burk plot (l/v vs l/s) and (D) Eadie-Hofstee plot (v vs v/s). The program is written such that the operator can select that form of graphical display that is thought to be most appropriate. Units of v and s are U/ml and mM, respectively.

zyme syringe, any variation in either the measured substrate concentration or the observed rate would be reflected in variations in the amount of solution delivered to the mixer. For an experiment with 18 rates, in which the AA was 0.05 in 20 s, the coefficient of variation in both the rate and the substrate concentration was below 1%. Coefficients of variation ranged between 0.47 and 1.7% for three repetitions of the experiment.

Inhibitor Studies Inhibitors can be examined in two ways: either the inhibitor is varied at fixed substrate concentration or the inhibitor is fixed while the substrate is varied. Using 2. mobilis ADH-2 (cobalt substituted), the K,,, for NAD+ and the Ki for NADH were measured (Fig. 7). Each determination was performed in duplicate; lines in Fig. 7 are drawn

through the intercepts given by the K,,, and V,, calculations on each set of data. Under these conditions, the K,,, for NAD+ is 40 + 4 PM and the Ki for NADH is 10 + 1 PM. Using 2. mobilis ADI-I-1 the K, for ethanol was first determined to be 4.0 mM at pH 10. The inhibitor trifluoroethanol, which is competitive with ethanol, was titrated into the assay mixture. The solution containing 10 mM inhibitor and 4 mM ethanol was placed in the substrate syringe. Enzyme in the enzyme syringe contained 5 mM NAD+. The mixing chambers were filled with 4 mM ethanol instead of water, thus giving the highest rate at the first pulse with 2 mM ethanol but no inhibitor present in the flow cell. The inhibitor titration, v vs [I], and l/v against [I] plots (Fig. 8) illustrate the high quality of the data obtained to provide an accurate measurement of the Ki of trifluoroethanol, 0.62 mM.

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greatly in their K,,, values, requiring widely different substrate concentration ranges to determine their Michaelis-Menten kinetic parameters. Concentrations of 20 and 0.2 mM, respectively, were used in the substrate syringe, and carboxypeptidase A was added to the enzyme syringe. Each solution was made up in 50 mM Hepes, pH 7.5, 0.1 M FIG. 7. Inhibition plots for Z. mobilis ADH isozyme 2 NaCl; this buffer was used to initially fill the using NADH as competitive inhibitor of NAD’. NAD+ chambers. Rhodamine B was added to the dissolved in water was the varied substrate in the sub- substrate solutions to give a l.OA reading at strate drive syringe. In addition to enzyme, the enzyme 550 nm. The Lineweaver-Burk plots (Fig. 9) syringe contained the indicated different fixed concenand provide Mitrations (X2) of NADH and ethanol at 100 mM, pH 9.5. show excellent linearity Lines were drawn according to calculated values of K,,, chaelis-Menten parameters for Fa-Phe-Phe and V,, for each run (done in duplicate as indicated by which are identical (K, = 100 PM, k,,, the paired lines at each NADH concentration). K,,, for = 40,000 min-‘) to those obtained preNAD+ = 40 pM. Right: Plot of the apparent K,,, against viously by manual methods (5). NADH concentration giving K, for NADH of 10 f 1 PM. Enzymatically Carboxypeptidase

A-Catalyzed

Hydrolysis

In order to apply the dilution analyzer to a more difficult analysis system and another enzyme, the kinetics of hydrolysis of two furanacrylolyl-substituted peptide substrates of bovine carboxypeptidase A were studied. Hydrolysis of these substrates results in a decrease in absorbance between 345 and 360 nm, producing an approximate 50% decrease in the initial absorption on complete conversion to products. The two substrates examined, Fa-Gly-Phe and Fa-Phe-Phe, differ

Coupled Assay

To demonstrate the effectiveness of the system in a coupled enzyme assay, pyruvate kinase was chosen. Lactate dehydrogenase (10 units ml-‘) was the coupling enzyme, with NADH at 50 HM. A 10-s pause was programmed to eliminate the lag period from the measurements, which were taken in the subsequent 20 s. Because the K, values for each substrate are low, the largest AAX4~ was kept below 0.03 to minimize substrate consumption. Phosphoenolpyruvate was the variable substrate, and nine different fixed

FIG. 8. Inhibition plots for Z. mobilis ADH isozyme 1 in which the inhibitor trifluoroethanol is varied at constant substrate concentration, 2.4 mM NAD+ and 2 mM ethanol. (A) Rate vs inhibitor concentration. (B) Reciprocal rate vs inhibitor concentration that provides a value for K, of 0.62 mM for competitive inhibition.

266

SCOPES AND HOLMQUIST

FIG. 9. Lineweaver-Burk plots obtained for the hydrolysis of Fa-Gly-L-Phe (A) and Fa-L-Phe-L-Phe (B) by bovine carboxypeptidase A. The reactions were monitored at 354 and 346 nm, respectively, and were followed for 30 s over which time the maximal AA was 0.016 with a background of 0.21 A. Units of s are mM and Y are A/30 s.

concentrations of ADP were used. Assuming a rapid equilibrium random mechanism, values for Kia, K,, Kib, and Kb (where a = ADP, b = phosphoenolpyruvate) were determined to be 20,65, 15, and 44 PM, respectively (Table 1). A separate determination of K, (by varying ADP at 1 mM phosphoenolpyruvate) gave a value of 58 PM. These results were all obtained in a 3-h period using the same solutions throughout. pH-Rate

Profiles

Instead of using a dye to indicate substrate concentration, a pH-indicator dye was used at constant concentration to provide a measurement of the pH in the cell based on the absorbance of one form of the dye. Starting at high pH, the indicator dye is gradually titrated with acid or buffer to cover the appropriate range. One indicator dye can be used to cover about 1 pH unit on either side of its pK,. Using bromthymol blue (pK, = 7.1), we determined the pH dependence between pH 6.1 and 8.2 of human &3, ADH and Z. mobilis ADH-2 under saturating substrate conditions (100 mM ethanol). The enzyme syringe contained all reagents except one substrate, plus bromthymol blue, in Tritine buffer at pH 8.4. The substrate syringe contained the ethanol in a mixture of 0.1 M Mes and 0.1 M Mops as titrating acid. The mixing chambers also contained ethanol in

water. Rates were determined by measurements at 340 nm and the pH was calculated from the absorbance at 6 18 nm, according to the Henderson-Hasselbalch equation. (The alkaline form of bromthymol blue has a peak at 6 18 nm; the acidic form does not absorb at this wavelength.) Data for human liver alcohol dehydrogenase isozyme ~$3~and for Z. mobilis alcohol dehydrogenase- 1 are shown in Fig. 10. A more extensive pH range could be covered if a mixture of indicators were used, e.g., bromthymol blue plus phenolphthalein. TABLE RESULTS

OF KINETIC mobilis P~RUVATE DEHYDR~GENASE

[.==‘I (PM) 500 333 250 150 100 70 50 50 35

1

EXPERIMENTS ON Zymomonas KINASE, USING LACFATE AS COUPLING ENZYME’

K”P (if)

Kl!L W’)

33 34 37 29 35 23 25 23 20

266 260 253 196 184 158 127 120 82

’ Rate measurements were made between 10 and 30 s alter mixing. Each determination of Km and V=$ represents 15 data points. Conditions: pH 6.5, K-Mes buffer, [I] = 0.07, [Mg*‘] = 5 mM, 25°C.

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FIG. 10. Plots of the rate against pH for ethanol oxidation by (A) human ab, ADH and (B) Z. mobilis ADH isozyme 2.

DISCUSSION

This simple apparatus was designed to mix enzyme automatically with successively incremented substrate concentrations in a way that eliminates manual pipetting and enables rate determinations to be initiated within 1 or 2 s. It also enables rate determinations to be made within a short time, typically lo-20 s per rate, with a sensitivity at least as high as manual procedures. Indeed, the amount of enzyme consumed in carrying out assays at 15 different substrate concentrations is less than would be used in the same number of individual l-ml manual assays. The system uses most of the sample to wash out previous sample, and rate measurements are actually made on only 8 ~1. Less substrate is also required. Once the substrate solution, containing dye to monitor dilution of the substrate, and enzyme solution, containing any other substrates or cofactors, are placed in the syringes, no further manual manipulations are necessary. Values for K,,,and V,,, are calculated automatically; the total process takes from 5 to 15 min, depending on the selected measurement times. We have shown that the system is very flexible within the limits of the spectrophotometric measurements and can be programmed to carry out inhibition constant determinations, pH-rate profiles, and other titration experiments that can be followed by spectral changes. For detailed studies of the kinetic parameters of enzymes, such as their variation with

pH, this instrument will considerably simplify and accelerate determinations. For instance, it would be possible to carry out measurements of both V,,, and K,,, at 0.02 pH increments over a total pH range of 3-4 units in a few hours, using the same reagents and enzyme sample throughout. Thus, detailed kinetic examinations of the increasing number of modified enzymes created by protein engineering could be carried out with ease. This, of course, assumes that a continuous spectrophotometric assay is available. The use of a dye in the substrate solution provides a number of advantages. Primarily, it allows a direct measurement of the dilution of the substrate in the actual solution being measured in the flow cell, concurrent with the rate measurements. This minimizes any errors encountered in routine cuvette assays, where one or more pipettings are required to vary substrate concentration and to initiate reactions by addition of enzyme. The primary dye used here, rhodamine B, has a high molar absorptivity that enables the use of very small concentrations. The highest concentration present in our assays, 3 PM, has no effect on any of the reactions studied. Alternative indicator dyes could be used in cases where the absorbance of rhodamine B, which occurs in the region 480 to 590 nm, would interfere with the spectral rate measurements. Dye incorporation also makes the design of the instrument very flexible, in the sense that the volume of the chambers and associated flow channels need not be

268

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precision machined to exacting specifications. Indeed, fabrication is quite simple, requiring only modest skills and a few tools. The volumes of the various parts control only the fine shape of the substrate dilution gradient. The dead volume of the design shown, i.e., the volume from the entrance to the mixing channels to the output of the cell, is 170 ~1, of which approximately 100 ~1 is for the mixing channels and 60 ~1 is for the tubing connecting the chamber block to the flow cell. However, slightly greater than 170 ~1 per pulse is required to fully flush out the sample from the previous pulse, due to laminar flow. Thus, when 200 ~1 of dye solution, with an absorbance of 1, was flushed through the system containing water, the absorbance in the cell was brought to 0.85. Nevertheless, it was found that good results were obtained with pulses as small as 200 ~1. These volumes can be reduced considerably by using finer mixing channels. It should be emphasized that the apparatus described is inherently quite tolerant of solution carryover, since a constant amount of enzyme is injected at each pulse and the substrate concentration varies only slightly between pulses. In the design presented, no attempt has been made to provide thermostatting of either the chamber block or the flow cell; all data presented have been obtained near room temperature, 23-25°C as measured with a microthermocouple attached to the flow cell. Temperature control could be achieved by channeling the chamber block and combining the flow cell and chambers into a slightly larger block, then thermostatting with flowing water. The entire assembly would be placed within the sample compartment of the Hewlett-Packard 845 1A, a design currently in development. An alternative procedure particularly suitable for substrates of low solubility and/or high expense is to fill the mixing chamber(s) with substrate and the syringe (S) (see Fig. 1) with water or buffer. Thus, during the run, the first points are at the highest substrate

concentrations, which then decrease during subsequent pulses. This mode of operation has provided data comparable to that in which substrate is included in the syringe (S). While the current system employs the programmable linear cell transport to drive the syringes, the torque provided by the small stepping motor is inadequate to drive larger syringes, which would give more flexibility in pulse programming and volume ranges. It would be a simple matter to incorporate a more powerful linear stepper motor drive. In conclusion, the novel incorporation of a sealed dilution chamber in the flow device described here, in combination with the use of the diode array spectrophotometer, has provided an extremely useful device which accurately, rapidly, and automatically carries out analyses requiring the systematic variation of the concentration of one component. We have documented its flexibility in enzymology in determining kinetic and inhibition constants, as well as pH profiles. Extension of this principle to other methods, for example, stopped-flow analysis, can be expected to provide an added degree of both speed and accuracy in data acquisition. ACKNOWLEDGMENTS This work was supported by a grant from the Samuel Bronfman Foundation, Inc., with funds provided by Joseph E. Seagram and Sons, Inc.

REFERENCES 1. Wagner, F. W., Burger, A. R., and Vallee, B. L. (1983) Biochemistry 22, 1857-l 863. 2. Ditlow, C. C., Holmquist, B., Morelock, M. M., and Vallee, B. L. (1984) Biochemistry 23,6363-6368. 3. Pawluk, A., Scopes, R. K., and Gtiffiths-Smith, K. ( 1986) Biochem. J. 238,275-28 1. 4. Neale, A. D., Scopes, R. K., and Kelly, J. M. (1986) Eur. J. Biochem. 154, 119-124. 5. Riordan, J. F., and Holmquist, B. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H. O., Ed.), Vol. 5, 3rd ed., pp. 55-60, Verlag Chemie, Weinheim. 6. Holmquist, B., and Vallee, B. L. (1979) Proc. N&Z. Acad. Sci. USA 76,62 16-6220. 7. Cleland, W. W. (1979) in Methods in Enzymology (Purich, D. L., Ed.), Vol. 63, pp. 103-138, Academic Press, New York.