A radiometric assay for HIV-1 protease

ANALYTICALBIOCHEMISTRY

l&$408-415

(1990)

A Radiometric Assay for HIV-1 Protease Lawrence J. Richard Departments SmithKline

Received

J. Hyland,*,i Brian D. Dayton,* Michael L. Moore,? A. Y. L. Shu,$ Heys,$ and Thomas D. Meek* of *Medicinal Chemistry, fPeptide Chemistry, ana’$Radiochemistry, BeechamPharmaceuticals,King of Prussia, Pennsylvania 19406

February

23,199O

A rapid, high-throughput radiometric assay for HIV1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-l protease on the radiolabeled form of a heptapeptide substrate, [tyrosyZ-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-ValNH%, which is based on the pl7-p24 cleavage site found in the viral polyprotein substrate Pr5Y”. Specific cleavage of this uncharged heptapeptide substrate by HIV- I protease releases the anionic product [ tyrosyl3,5-‘H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AGl-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources. 0 1990 Academic Press, Inc.

The proteolytic processing of the polyprotein products of the gag and pal genes of the human immunodeficiency virus (HIV-l; (1)) is accomplished by a virally encoded aspartic protease (2). This enzyme has been shown to be crucial to the maturation, replication, and infection-competence of HIV-l (3-6). As a result, the development of specific inhibitors of HIV-l protease has received intense recent interest as a potential therapeutic strategy for the treatment of AIDS and related diseases. Toward this goal, HIV-l protease has been expressed in Escherichia coli (7) and has been purified from bacterial lysates (8). Characterization of this enzyme in several laboratories has shown that a number of oligo‘To whom dressed.

reprint

requests

and

correspondence

should

be ad-

peptides which are based on the known cleavage sequences found within the HIV-l polyproteins are substrates for the protease (9-18). The most thoroughly studied of these substrates contain a sequence which is similar to the cleavage site at the p17-p24 junction of Pr55g”g, Ser-Gln-Asn-Tyr * Pro-Ile-Val(l9). To date, kinetic assays for HIV-l protease have relied on either HPLC or spectrophotometric analysis of peptidolytic products. Both types of assay are adequate for kinetic analysis conducted on a small scale, yet suffer from several disadvantages which restrict their use in a facile, high-throughput assay for HIV-l protease. HPLC assays are time-consuming and are limited to singlepoint sampling, while the sparingly soluble oligopeptides used as spectrophotometric substrates produce relatively small chromophoric changes (17,lS). We have developed a peptidolytic assay which is based on the quantification of a radiolabeled oligopeptide product, separated from substrate by ion-exchange chromatography conducted in a 96-well filtration plate. This methodology allows the measurement of the peptidolytic activity of HIV-l protease to be conducted on a large array of samples simultaneously. By virtue of its radiometric design, the resulting data are not subject to background interferences from contaminants which would complicate optical detection in HPLC and spectrophotometric assays. MATERIALS

AND

METHODS

HIV-l protease. Recombinant HIV-l protease was obtained from the PRO4 expression vector in E. coli strain AR58 as described (7,8). The protease was purified to >90% homogeneity by the method of Strickler (8), and the purified enzyme was stored at -20°C in 20 mM Tris-HCl (pH 7.5), 1 mM DTT,’ 1 InM EDTA, 200 2 Abbreviations used: Asn-Tyr-Pro-Val-Val-NH2, 2-(N-morpbolino)ethanesulfonic N,N-dimethylformamide;

AcSQNY * PVV-NH2, N-acetyl-Ser-Glnwhere * signifies the cleavage site; Mes, acid; DTT, dithiothreitol; DMF, DMSO, dimethyl sulfoxide. 0003-2697/90

408 All

Copyright 0 1990 rights of reproduction

by

Academic in any

form

$3.00 Inc. reserved.

Press,

RADIOMETRIC

ASSAY FOR HIV-1

PROTEASE

409

mM NaCl, 40% glycerol (enzyme storage buffer) at protein concentrations of lo-30 pg/ml. Protease concentrations were measured as described by the chromatographic method of Strickler (8) and by using the BCA protein reagent of Pierce Chemical Co.

Peptide synthesis. Ac-Ser-Gln-Asn-Tyr-Pro-ValVal-NH2 was prepared by solid-phase peptide synthesis on benzhydrylamine resin and purified as described (lo), colloctlon rack and its structure was confirmed by amino acid analysis 1 Y and fast atom bombardment mass spectrometry. The FIG. 1. Schematic representation of filtration plate manifold for tritiated form of this peptide was prepared by catalytic conducting ion-exchange chromatographic steps of the radiometric astritiation of the (tyrosyl-3,5-I,)-Ac-Ser-Gln-Asn-Tyrsay in a simultaneous g&sample array. Quenched samples are added to the wells of the filtration plate which contain minicolumns of AGIPro-Val-Val-NHe. Iodination was performed as follows: wash the resin 16 mg Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 was X8 resin. Positive air pressure is then used to uniformly with various eluants; the corresponding eluates are collected in test dissolved in 10 ml 0.2 M Na,HPO, (pH 7.0) containing tubes contained within a rack which is complementary to the filtration 5.9 mM sodium iodide. Freshly prepared 5.9 mM chloraplate. mine-T (10 ml) was added, and the mixture was incubated for 60 min at 25°C. The reaction was quenched by plete enzymatic digestion using reaction conditions as the addition of 10 ml 5.9 mM L-cysteine in 0.2 M previously described (10). The product was purified by Na2HP04 (pH 7.0). The iodinated product was purified by HPLC on a semipreparative Altex C-18 column (10 preparative HPLC, lyophilized, and redissolved in wax 150 mm, 5 pm) using a mobile phase (4 ml/min) con- ter. Spectral comparison of the isolated compound and resulted in identical prosisting of 20% acetonitrile in 0.1% trifluoroacetic acid the authentic AcSQNY-CO; files, and HPLC coinjection displayed an identical chro(10 min), followed by a linear gradient of 20-30% acetomatographic profile. nitrile in 0.1% trifluoroacetic acid for 20 min with detection at 220 nm. Eight milligrams of the iodinated prodOther materials. Pepstatin A was purchased from uct (38%), which eluted at 23-24 min, was recovered as Sigma Chemical Co. (St. Louis, MO). Dowex AGl-X8 a solid following lyophilization. The molecular weight of anion-exchange resin (100-200 mesh, formate form; 1.2 the product as determined by FAB/MS was consistent meq/ml of wet resin) and Bio-Beads SX-8 were obtained with that expected for the diiodinated heptapeptide from Bio-Rad Laboratories (Richmond, CA). Filtration (m/e 1099). plates (catalog No. 123) were purchased from V&P SciA 2.47-mg portion of (tyrosyl-3,5-I,)-Ac-Ser-Glnentific (San Diego, CA). The positive pressure manifold Asn-Tyr-Pro-Val-Val-NH2 was dissolved in 1 ml of 5: (9 X 20 cm) was designed and constructed in-house out 95 triethylamine:DMF, and 1.45 mg of 10% Pd/C was of polypropylene and made to fit snugly over the filtraadded. The mixture was stirred under 5 Ci of tritium gas tion plate (Fig. 1). Collection tubes (8 X 40 mm polystyat 25°C for 2 h. After removal of unreacted tritium gas, rene, 1.2 ml; catalog No. 265270) and collection plates the catalyst was separated by filtration and the solvent (catalog No. 265272) were purchased from Beckman Inwas removed from the filtrate by static vacuum transfer. struments (Fullerton, CA). All other reagents were of Labile tritium was removed by twice adding and lyophianalytical grade or of the highest available purity. lizing away 2-ml portions of 90% aqueous methanol. Enzyme assays. Assay of the peptidolysis of unlaThe residue was purified by semipreparative HPLC (Albeled AcSQNYPVV-NH, by HIV-l protease using tex Ultrasphere C-18) using a mobile phase (2.5 ml/ HPLC was as described (10). The concentration of AcSmin) consisting of 18% acetonitrile in 0.2% trifluoroQNYPVV-NH, within stock solutions was determined acetic acid, resulting in 52.5 mCi of [tyrosyl-3,5-3H]Acfrom analysis of the uv spectrum, based on a value of Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2(13H]AcSQNYf 275nm= 1420 M-’ cm-l for the tyrosyl residue. For the PVV-NH2). This product was stored as a solution in assay of the tritiated heptapeptide, reaction mixtures 90% aqueous ethanol at -80°C. The radiochemical pu- (20 ~1) containing 50 IIIM Mes, pH 6.0, 1 mM EDTA, 1 rity was found to be 98.6% (two analytical Altex Ul- mM DTT, 0.2 M NaCl, 0.1% Triton X-100 (Buffer A), trasphere ODS columns in series, mobile phase as and 0.5-5 mM [3H]AcSQNYPVV-NH2 (4.5-5.3 mCj/ above), and the specific activity by calculation from the mmol) were pipetted into the wells of a Linbro V-bottom mass (HPLC) and radioactivity (liquid scintillation 96-well plate which was then preincubated in a water counting) was 52 Ci/mmol. The tritiated product bath at 37°C for several minutes. Reactions were initiated by the addition of 3-5 ng of HIV-l protease (5 pl), [3H]Ac-Ser-Gln-Asn-Tyr-CO; was prepared by diluting the radiolabeled substrate with unlabeled substrate and following incubation at 37°C for 10 min, reactions to a specific activity of 4.5 mCi/mmol, followed by com- were quenched by the addition of 10 vol of ice-cold water

410

HYLAND

(or ice-cold 10 mM Tris, pH 8.5), and the plate was placed on ice for 10 min. This reaction quench treatment was determined as adequate enough to fully inhibit further enzymatic activity. Control samples, in which enzyme was added to the reaction mixtures following quenching, were treated in an otherwise identical manner, and provided a background subtraction from each sample. Separation of the radiolabeled product from the substrate was achieved on an array of “minicolumns” of Dowex AGl-X8 (formate), which were prepared in a 96well filtration plate (Fig. 1) as follows: AGl-X8 resin was prepared in a 1:l (v/v) suspension in water just prior to use. Aliquots (250 ~1) of this suspension were loaded into each well of the 96-well filtration plate with a multichannel pipettor, and the filtration plate was placed directly on top of collection tubes set in the matching 96-well tray (Beckman). The quenched reaction mixtures were then added to this minicolumn array, and the positive pressure manifold was set firmly on top of the plate. Low-pressure air (ca. 30 psi at the inlet) was then gently applied, and the unreacted [3H]AcSQNYPVV-NH2 was eluted with 2 ml of deionized water which was evenly pushed through each well of the filtration plate and collected. The entire filtration plate was then transferred onto a new set of collection tubes, and the radiolabeled product, [3H]AcSQNY-CO;, was eluted by washing the minicolumns with 2 ml of 7 N formic acid using positive air pressure. The eluants were collected in a 96-tube array as before. The tritium content within the fractions containing separately recovered peptidolytic substrate and product was then determined by adding the eluants to vials containing Beckman Ready-Protein scintillation cocktail (20 ml total). The sample vials were counted on a Beckman Model 5801 liquid scintillation counter and the results were expressed in dpm. To reduce the error resulting from small variations in applied sample volumes or in elution of the assay samples, initial rates were calculated from the ratio (dpm product/(dpm product + dpm substrate)) of recovered radioactivity. Enzyme kinetics. Kinetic constants were determined from initial rate data obtained over linear time courses at variable concentrations of [3H]AcSQNYPVV-NH2 by fitting to the Michealis-Menten equation,

VA u=K+A,

PI

using the FORTRAN program (HYPER) of Cleland (20). In Eq. [l], u is the initial rate, V is the maximal velocity, A is the substrate concentration, and K is the Michaelis constant. Inhibition constants were determined using the method of Dixon (21) at a single substrate concentration (1.0 mM), assuming competitive inhibition.

ET

AL.

Kinetic parameters were also determined at pH 3-7 using a mixed buffer of 50 mM each of glycine, acetic acid, Mes, and Tris, 1 mM EDTA, 0.2 M NaCl, 1 mM DTT, 0.1% Triton X-100 (Buffer B). After the desired pH value was attained, the ionic strength of each solution was adjusted to constant conductance by the addition of small aliquots of 3 M NaCl. Replicate reaction mixtures (50 ~1) containing Buffer B, 0.5-5 mM [3H]AcSQNYPVV-NH2 (10,000 dpm/nmol) were preincubated for several minutes at 37°C and the reaction was initiated by the addition of 40-150 ng purified HIV-l protease depending on the pH for that sample. Samples were quenched at both 5 and 10 min as described above. Upon preincubation at various values of pH followed by assay at pH 6, the stability of HIV-l protease activity was limited to a pH range of 3-7. Kinetic constants (V/ K, V) obtained from a fitting of these data to Eq. [l] at each value of pH were then fitted to either logy = log

c 1 + H/K,

+ K,/H

Bl

for a bell-shaped pH profile where the value of y decreases with slopes asymptotically approaching 1 and -1 at low and high pH, respectively, or logy=log

(YL+

YH&/H)

1 + K,/H for a wave-shaped pH profile where protonation of a single group results in a shift of y to a lower constant value. In Eqs. [2] and [3], y is the kinetic parameter V or V/K, c is the pH-independent value of y, YL and Yn are the values of y at low and high pH values, respectively, H is the concentration of hydrogen ion, and Kl , K2, and K3 are the dissociation constants of the ionizing groups. RESULTS

AND

Characterization

DISCUSSION

of the Radiometric

Assay

The ability of the anion-exchange resin AGl-X8 (formate) to separate [3H]AcSQNY-C0; from unreacted [3H]AcSQNYPVV-NH2 is demonstrated in Fig. 2. Two “mock” enzyme assays representing 0% and 100% peptidolysis of [3H]AcSQNYPVV-NH2 were applied in triplicate to identically prepared minicolumns of AGl-X8 (formate). Greater than 97% of the substrate [3H]AcSQNYPVV-NH, was eluted from the resin by six column volumes (0.75 ml) of water. Extended washings, for up to 20 column volumes of water, typically resulted in the elution of less than 3% of the remaining bound [3H]AcSQNYPVV-NH2 . Upon application of the carboxylate product [3H]AcSQNY-C0;, the radiolabel was retained by the anion-exchange resin following water washing up to 20 column volumes (2.5 ml). Elution with

RADIOMETRIC

7N Formic

Acid

ASSAY

P I’ \ /

‘,

:

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:

‘, .-

; I’

--a

\ ‘\ \

, f’ 0

2

4

h

6

a

10

Fraction

FIG. 2.

Elution of radiolabeled substrate and product from AGl-X8 minicolumns using the filtration plate manifold apparatus. Three 20. ~1 replicates each containing Buffer A, enzyme storage buffer, and either 1 mM [3H]AcSQNYPVV-NH, or 0.2 mM (3H]AcSQNY-C0, were prepared, and approximately 40000 dpm of each sample was applied to a 0.125-ml minicolumn of AGl-X8 (formate). Each column was successively washed with 0.5 ml of water and 0.75 ml of 7 N formic acid (arrow), and 0.125-ml fractions were collected and counted as described under Materials and Methods (dpms of [‘H]AcSQNYPVVNH,, solid squares, solid line; [3H]AcSQNY -CO;, solid circles, dashed line). The average of the three samples is shown.

8-12 column volumes (l-l.5 ml) of 7 N formic acid resulted in the specific elution of all of the bound radiolabeled product from the AGl-X8 minicolumns. These elution characteristics for the substrate were consistent over a broad concentration range (O-02-5 mM). On the basis of these results, we used for our standard elution conditions 2 ml of water to elute unreacted substrate, followed by 2 ml 7 N formic acid to elute product. This separation method was then applied to a reaction mixture in which peptidolysis of 0.5 mM [3H]AcSQNYPVV-NH2 (50,000 dpm total) by purified HIV1 protease had proceeded to approximately 19% (as shown by monitoring a replicate, unlabeled sample by HPLC analysis). A total of 37,860 dpm and 7540 dpm were collected in the water and formic acid washes, respectively, indicating 17% conversion of substrate to product based on recovered radioactivity (91% of the applied sample). To validate that substrate and product had been completely separated by this procedure, the water and formic acid eluants were collected, lyophilized, and redissolved in a minimal volume of water. These samples were then analyzed separately by reversed-phase HPLC using chromatographic conditions described under Materials and Methods, and 0.75-ml fractions were collected and counted. All of the recovered tritium was associated with a single chromatographic peak of retention time and spectral characteristics identical to that of either authentic AcSQNYPVVNH2 or AcSQNY-CO;. These results indicate that the

FOR

HIV-1

411

PROTEASE

radiolabeled substrate and product are well separated by ion-exchange chromatography using this stepwise elution protocol. Typically, recoveries of the total radioactivity applied, water and formic acid washes, were nearly quantitative. The data in Table 1 represent sampling at various time points of an enzymatic reaction mixture in which the peptidolysis of 5 mM [3H]AcSQNYPVV-NH2 had proceeded from O-80% in 180 min. The progress of the enzyme-catalyzed peptidolysis is evident in the time-dependent enrichment of radioactivity in the formic acid washes and indicates that the separation of product from substrate is well behaved regardless of the extent of reaction. The binding capacity of the minicolumns of AGl-X8 (formate) typically used in this assay was evaluated as follows: triplicate lo-p1 solutions containing Buffer A and 50 nmol of purified t3H]AcSQNY -CO, (10,000 dpm/ nmol; 5 mM) were applied to 0.125-ml columns of AGlX8 (formate). The minicolumns were then individually equilibrated with and washed with similar “quenched reaction mixtures” containing either unlabeled product (5 mM AcSQNY -CO, ) or unlabeled substrate (5 mM AcSQNYPVV-NH2), and radiolabel in the eluates was quantified. Less than 1% of the applied radioactivity was detected upon repeated equilibration with either the unlabeled product or substrate. However, the bound, labeled product could be eluted quantitatively upon application of 2 ml 7 N formic acid, thereby demonstrating again that the interaction of bound [3H]AcSQNY-C0,

TABLE Recovery

Reaction time (min)

Water eluate (dpm)

Formic acid eluate kbm)

12 15 30 60 120

573,897 570,350 506,450 356,710 222,680

5,290 7,840 10,040 25,105 20,830 29,569 32,330 65,610 212,170 349,210

180

115,600

461,980

0

3 5 7 10

584,440 579,240 587,690 568,210

of Radioactivity of Enzymatic

575,110

1 during Reaction”

Fractional conversion to product

0.000 0.0044 0.0078 0.033 0.026 0.040 0.045 0.105

0.360 0.600 0.790

Progress

Total b

recovery kbd

589,730 (99.8%) 587,100(99.3%) 597,730 (101%) 593,310 (100%) 595,930 (101%) 603,470(102%) 602,680(102%) 572,060(96.8%) 568,870 (96.3%) 571,893 (96.8%) 577,581(97.7%)

’ Incubation of 5 mM [3H]AcSQNYPVV-NH2 in 10 ~1 Buffer A containing 7 ng HIV-l protease at 37°C with quenching at the indicated times. ’ Product formation is dpm formic acid eluate/total dpm recovered. The zero time point of formic acid eluate is subtracted from subsequent values. Total averaged dpm applied from each sample was 591,000. Results are the averages of duplicate samples.

412

HYLAND

ET

AL.

with the anion-exchange resin is specific and well-behaved. The capacity of the minicolumns is sufficient to bind all of the [3H]AcSQNY-CO; that would result from the total cleavage of AcSQNYPVV-NH, at a concentration (50 mM) well above those which are typically used. These results indicate that the 0.125-ml ion-exchange columns in this multisample assay format possess sufficient capacity to effectively retain all of the applied [3H]AcSQNY-CO; over the range of substrate concentrations used in these assays (0.02-5 mM). We found the formate form of AGl-X8 (100-200 mesh) to be the most suitable resin for the ion-exchange assay. The choice of AGl-X8 (formate) as the ion-exchange resin is based on the reported rapid exchange of the formate ion with monocarboxylic anions (27), and was found to be superior to the acetate and chloride forms of this resin. The ability of radiolabeled substrate or product to nonspecifically bind to the resin matrix was examined using a neutral resin of similar composition, Bio-Beads S-X8. No significant interaction of either product or substrate with the neutral resin was noted, both passed directly through the minicolumn upon washing with four column volumes of water. This result demonstrated a specific interaction of the anionic product with the functional diethylaminoethyl ligands of AGl-X8, with negligible interaction with its polystyrene backbone. In addition, a variety of eluants were compared on the basis of the efficiency of elution of the radiolabeled product (percentage recovery of identical amounts of bound [3H]AcSQNY-C0; in a constant volume of eluant). The eluants (7 N, unless otherwise noted) used and the corresponding relative percentage of product recovered were: acetic acid (88%), citric acid (l%), formic acid (loo%), hydrochloric acid (41%), phosphoric acid (21%), sulfuric acid (27%), potassium chloride (40%), and 5 N sodium chloride (40%). Of these, 7 N formic acid was the most efficient. Buffers composed of various mixtures of formic acid and ammonium formate afforded no improvement over 7 N formic acid. In order to monitor any interference by reaction mix components, 10% DMSO and 10% Triton X-100 were examined and found to be ineffective in removing bound radiolabeled product from the resin.

course of the peptidolysis of [3H]AcSQNYPVV-NHz as shown in Fig. 3A appeared to be linear for at least 9 min, corresponding to a fractional conversion of 20% of the substrate to product. Similar results have been observed for longer assay periods (20-30 min) in which the extent of peptidolysis was ~20%. The initial rate as measured from the slope of a plot of [3H]AcSQNY-CO; formed vs time was 23 f 3 nmol [3H]AcSQNY-CO; formed emin-’ . p8-l protease, and the corresponding y-intercept exceeded the background radioactivity in control samples subtraction by only 8%. These results indicate that within these experimental parameters (t < 10 min, product formation C 15-20% of initial substrate concentration), one can calculate an initial rate for the peptidolytic reaction from a single time point. We have observed few discrepencies between the initial rate obtained from the slope of a reaction time course and that measured from a single time point taken within the same range of experimental values. Furthermore, the near-zero y-intercept of the time course in Fig. 3A confirms that chilling and dilution by cold water is sufficient to quench the enzymatic reaction, as has been confirmed in timecourse studies in which quenching preceded the addition of radiolabeled substrate. The radiometric peptidolytic reaction was also linear with respect to enzyme concentration. Shown in Fig. 3B is a plot of the radiometric product formed at increasing amounts of protease (lo150 ng) during a fixed lo-min reaction period. For all data points, the extent of product formed was <19% of the initial substrate concentration.

Evaluation

Steady-state kinetics. Analysis of steady-state kinetic parameters of HIV- 1 protease using the radiometric assay yielded results which were nearly identical to those obtained from HPLC-based analyses. Shown in Fig. 4 is a double-reciprocal plot of initial rate data at variable concentrations of [3H]A~SQNYPVV-NHz, obtained from duplicate samples taken at a single time point. Fitting of these data to Eq. [ 1] resulted in the kinetic constants K = 5.3 f 0.6 mM, V/E, = 62 + 5 S-‘, and V/K. E, = 11.7 + 0.4 mM-’ s-l. These values were comparable to those obtained by HPLC analysis: K

as an Assay of Enzymatic

Activity

Dependence of reaction on time and enzyme concentration. Our previous characterization of the peptidolytic reaction of HIV-l protease demonstrated a linear time dependence of product formation at ~20% conversion in a 20-min incubation (10). These results indicated that the protease remained stable under reaction conditions which are identical to our present analysis. Similar linearity with time was observed for the radiometric assay over incubation periods of lo-20 min in which product formation had proceeded to 15-20%. A typical time

Reproducibility. In a study of the peptidolysis of [3H]AcSQNYPVV-NH2 at concentrations of 3-10 mM, initial rates were obtained from a single time point using the fractional formation of product (Table 2). The high reproducibility observed in these duplicate samples is reflected in the standard deviation of the calculated molefraction of product formation (0.2-5%). The precision with which the initial rates shown in Table 2 were measured is typical for the radiometric assay over a broad range of AcSQNYPVV-NH2 concentrations. Furthermore, these initial rate values compare favorably with those obtained for unlabeled AcSQNYPVV-NH2 by using HPLC to separate and quantify product from substrate.

RADIOMETRIC

ASSAY

FOR

HIV-l

413

PROTEASE

Minutes

Enzyme

(ng)

FIG. 3. (A) Time course of the HIV-l protease-catalyzed peptidolysis of 5 mM [3H]AcSQNYPVV-NH2 in Buffer A at 37°C. The reaction was initiated by the addition of 50 ng purified protease and quenched at the indicated times, and [3H]AcSQNY-CO; (in dpm) was separated and quantified as described under Materials and Methods. The line drawn through the experimental data points was obtained from a linear least squares fit (r = 0.9995; slope = 11,500 + 160 dpm/min; y-intercept = 800 dpm). (B) Dependence of the radiometric assay on enzyme concentration. Enzymatic reactions in mixtures (50 ~1) containing Buffer A and 5 mM [3H]AcSQNYPVV-NH2 at 37°C were initiated by the addition of 10.~1 aliquots containing O-150 ng purified HIV-l protease, and were quenched after 10 min. Linear regression of the averages (n = 3) resulted in the line drawn through the experimental points (r = 0.997; slope = 0.341 + 0.01 nmol/ng; y-intercept = -0.03).

= 7.5 mM, V/E, = 54 s-l, and V/K.E, = 7.2 mM-’ s-l (10). Similarly, a plot of l/u versus pepstatin A at 1 mM [3H]AcSQNYPVV-NH2 resulted in a Ki = 2.1 k 0.1 PM (assuming competitive inhibition), which is comparable to published values of 1.1 and 1.4 PM obtained by HPLC (11,22). Kinetic analysis, using HPLC!, of many of the oligopeptide substates of HIV-l protease have been complicated by limited solubilities and by high Michaelis constants (11,15,18). An advantage of the radiometric assay is the ability to measure reaction rates at micromolar concentrations of AcSQNYPVV-NH, by increasing the specific activity of the substrate. Reliable data could be obtained at 0.02-5.0 mM [3H]AcSQNYPVVNH2 by varying the substrate’s specific activity over a

TABLE

Reproducibility

loo-fold range. A plot of initial rate vs substrate concentration conformed to typical Michaelis-Menten behavior over this concentration range, and kinetic constants determined from double-reciprocal analysis of these data were consistent with those obtained at millimolar substrate concentrations: K = 3.4 f 0.8 mM and V/E, = 65.9 Ifr 11 s-l. These results demonstrate that kinetic parameters obtained solely at millimolar concentrations of AcSQNYPVV-NH2 reflect the continuity of kinetic behavior observed over a much broader concentration range. The pH rate behavior of HIV-1 protease was also examined using this assay method. Values of V/K and V were determined as above over a pH range of 3-7 using

2

of Assay at Variable Substrate Concentrations” [3H]AcSQNYPVV-NH2

Water Formic Fractional

eluate

(dpm)

acid eluate

(dpm)

conversion

to product

Averaged values Initial rate (nmol AcSQNY-CO,/min) (LAt 37”C, in 10 ~1 Buffer dpm formic acid eluate/total

concentration

3mM

5mM

9mM

10 mM

290,730 291,500 40,180 40,430 0.1214 0.1218 0.1216 t 0.0003

525,200 526,620 59,060 54,450 0.1011 0.0937 0.0974 + 0.0052

787,700 781,810 65,020 69,470 0.0762 0.0816 0.0789 k 0.0038

876,610 873,790 76,130 76,170 0.0799 0.0802 0.0800 z!z 0.0002

1.824

2.435

3.55

4.00

f 0.004

A (pH 6.0) containing 9 ng HIV-1 protease; reaction recovered dpm. Initial rate is calculated as (product

+ 0.160 was quenched fraction)(nmol

f 0.17

after 10 min. Fractional substrate)/time.

conversion

f 0.01 to product

is

414

HYLAND

a single mixed buffer. Prior examination demonstrated that the protease activity remained stable following preincubation at each pH, and that the time course of peptidolysis at each pH remained linear well beyond the assay period. The pH-dependence of V/K and V are plotted logarithmically in Fig. 5. The plot of log V/K vs pH is a bell-shaped curve in which V/K decreases with slopes of 1 and -1 at low and high pH, respectively. Values of pK, = 3.3 +- 0.09 and pK, = 6.9 t- 0.18 were obtained from fitting of these data to Eq. [2]. Since V/K is a measure of the enzymatic reaction rate at very dilute substrate and represents the rate constants up to and including the first irreversible reaction step, this plot indicates that the catalytic form of the enzyme to which the uncharged hexapeptide substrate binds contains both an unprotonated and a protonated group of pK = 3.3 and 6.9, respectively (23). This curve is similar to that reported for the aspartic protease penicillopepsin (24). The pK values of both of these catalytic residues are within a range which denotes carboxylic groups. In addition, an unprotonated group of pK = 3.8 has been demonstrated in the inactivation of HIV-l protease by 1,2epoxy-3-(4-nitrophenoxy)propane (9), a reagent which esterifies Asp-32 in the active site of porcine pepsin (25). The profile of log V vs pH, fitted to Eq. [3], is defined by a “wave”, indicating that protonation of a residue of pK = 4.6 * 0.09 decreases, but does not abolish, the enzymatic reaction at saturating substrate concentration. Such a profile is unusual for the pH dependence of V,

ET

AL.

*.O( 1.6 1.2

/++t

0.8 t

1.8

3

4

5

6

7

PH

FIG.

5.

Variation of V/K and V with pH. Initial rates of [3H]AcSQNYPVV-NH, peptidolysis (l-10 mM) were measured in reaction mixtures (50 ~1) containing Buffer B adjusted to the indicated pH. 75 or 150 ng HIV-l protease were added at high and low pH, respectively. Values of V (squares) and V/K (circles) were obtained by fitting the data to Eq. [l]. The calculated curve drawn through the experimental points were obtained by fitting values for log V/K vs pH to Eq. [Z] and log Vvs pH to Eq. [3].

and suggests either a change in the rate-limiting step in the reaction mechanism or a conformational change of the enzyme upon protonation of the acidic residue, as has been suggested for similar kinetic behavior in the glucose-6-phosphate dehydrogenase from Leuconostoc

mesenteroides

(26).

SUMMARY

l/[[3H]AcSQNYPVV]

(mtd)

FIG. 4. Double-reciprocal plot of the initial velocity of HIV-l protease-catalyzed peptidolysis of [3H]AcSQNYPVV-NH2. The line drawn through the experimental points in the plot was obtained by fitting the data to Eq. [ 11. Reaction mixtures (40 ~1) at 37’C containing Buffer A (pH 6.0) and 0.5-4.0 mM [3H]AcSQNYPVV-NH2 (10,000 dpm/nmol) were initiated by the addition of 10 ng (8 ~1) enzyme and quenched after 10 min, and data were acquired and analyzed as described under Materials and Methods. Initial rate data were calculated from the averages of duplicate samples for each concentration of substrate. The fractional formation of [3H]AcSQNY-CO; in these samples was ~3% of the initial substrate concentration.

We have developed a reliable assay for HIV-l protease which uses a synthetic radiolabeled substrate. This sensitive and rapid radiometric assay is ideally suited for the detection of protease inhibitors within complex mixtures and for monitoring protease activity during its purification. Although the assay was developed primarily as a tool for identifying inhibitors of the HIV-l protease from natural product sources, we have demonstrated in this report that this method constitutes a robust assay for detailed kinetic analysis of the protease. Given the similarity in substrate preferences among the retroviral proteases, [3H]AcSQNYPVV-NH2 may also be useful in the development of assays for other members of this family of enzymes. Moreover, this approach could be expanded to include any protease in which an appropriate peptide substrate can be prepared in radiolabeled form. ACKNOWLEDGMENTS We thank Dr. James E. Strickler and Ms. Joselina Gorniak for gifts of the purified HIV-l protease, Drs. Terry A. Francis and Mark Levy for helpful discussions, and Dr. Brian W. Metcalf for support of this

RADIOMETRIC research. Supported Health Cooperative

ASSAY

in part (B.D.D.) by a National Drug Discovery grant (AI2484502).

Institutes

of

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