- Email: [email protected]
Enzyme-Coupled Assays for Proteases
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
241, 1–4 (1996)
0368
Enzyme-Coupled Assays for Proteases1 Brian E. Cathers and John V. Schloss2 Department of Medicinal Chemistry, 4070 Malott Hall, University of Kansas, Lawrence, Kansas 66045
Received November 30, 1995
MATERIALS AND METHODS We have developed a general strategy for assaying proteases that does not require the use of fluorogenic, chromogenic, or radiolabeled peptide substrates. The endoor exoproteolytic hydrolysis of simple peptides can be followed spectrophotometrically by coupling the proteolytic event via enzyme-catalyzed reactions to a chromogenic redox dye. The couple can be used directly to follow the action of carboxy or amino peptidases on peptide substrates or can be coupled by use of carboxy or amino peptidases to follow the action of endoproteases on peptide substrates that are blocked at the amino or carboxy terminus, respectively. Liberated amino acids are detected by use of amino acid oxidase, oxygen, horseradish peroxidase, and the redox dye 2,2*-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (e414 nm Å 36,000 M01 cm01). q 1996 Academic Press, Inc.
An assay strategy for proteases that employs simple, well-characterized, low-molecular-weight peptides composed of the 20 common amino acids would have great utility in characterizing the kinetic behavior of novel proteases. If such an assay were spectrophotometric-based and adaptable to a 96-well plate format, it would have the additional advantages of being quantitative, convenient, and useful for high-capacity screening of potential inhibitors. After exploring several potential enzyme-coupled assays for proteases, we have developed a protocol that meets the aforementioned criteria. Described below are the specifics of what, to the best of our knowledge, constitutes the first enzyme-coupled, spectrophotometric assay for proteases that employs simple nonchromophoric peptides. 1
The authors gratefully acknowledge financial support from the General Research Fund of the University of Kansas, the J. R. and Inez W. Jay Research Fund, the National Institutes of Health (GM07775), and the Burroughs Wellcome Co. We also thank Dr. Bruce Korant and Dr. Charles A. Kettner of the DuPont–Merck Pharmaceutical Co. for their generous gift of research materials. 2 To whom correspondence should be addressed. Fax: (913) 8645326. E-mail: [email protected].
Biochemicals L-Amino acid oxidase (Crotalus adamanteus EC 1.4.3.2), 2,2*-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),3 bovine carboxypeptidase A (EC 3.4.2.1), hippuryl-phenylalanine, horseradish peroxidase (EC 1.11.1.7), and porcine pepsin (EC 3.4.4.1) were purchased from Sigma Chemical Co. The tethered, single-polypeptide form of recombinant HIV protease, as prepared by the method described by Cheng et al. (1, 2), was a gift from Dr. Bruce Korant (DuPont– Merck Pharmaceuticals). The peptide substrate (ATHQVYPhe(NO2)VRKA) was a gift from Dr. Charles A. Kettner (DuPont–Merck Pharmaceuticals). The peptide substrate (AHQAFFVRKA-amide) was prepared by use of the DuPont RaMPS protocol with the Rapid Amide resin. Stock solutions of bovine carboxypeptidase A (1 mg/ml) were prepared by dissolving the aqueous suspension in 2.4 M LiCl/10 mM TrisrHCl (pH 7.5). Stocks of pepsin (5 mg/ml) were prepared by dissolving the lyophilized powder in sodium formate buffer (10 mM, pH 3.75). Horseradish peroxidase stocks (5–6 mg/ ml) and peptide substrate stocks (5–20 mM) were prepared as unbuffered aqueous solutions.
ABTS Coupled Assay The coupling enzymes, buffers, and reagents were employed at the following final concentrations: 0.1 M LiCl, 0.05 M TrisrHCl (pH 7.5), 2 mg/ml carboxypeptidase A, 70 mg/ml amino acid oxidase, 50 mg/ml horseradish peroxidase, and 0.8 mM ABTS. This coupling enzyme solution should be prepared at least 20 min before use and can be stored for up to 5 h at 257C. If carboxypeptidase A is omitted (to be added just prior to use), then the coupling solution is stable for up to 16 h at room temperature. Endoproteases were preincubated with peptide substrate at 257C for fixed periods of time in 20 to 50 mM 3 Abbreviation used: ABTS, 2,2*-azino-bis-(3-ethylbenzthiazoline6-sulfonic acid.
1
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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buffer (sodium formate, pH 3.75; potassium acetate, pH 4.75; MesrNaOH, pH 6.2; or TrisrHCl, pH 7.4) with various concentrations of KCl (0, 0.15, 0.38, or 0.45 M). Aliquots of the endoprotease assay solution were added to the coupling enzyme solution to a final volume of 1 ml in a cuvette or 0.25 ml in a 96-well plate. The increase in absorbance at 414 nm was monitored with a Shimadzu UVPC-2101 spectrophotometer or with a Molecular Devices THERMOmax microplate reader in kinetic mode using a 414-nm filter. Reaction rates were determined from the maximum absorbance obtained upon addition of the coupling enzyme solution. NADH Coupled Assay A modified version of the above assay that ‘‘chemically coupled’’ (nonenzymically) the oxidized ABTS to NADH was used initially for the assay of carboxypeptidase A. A catalytic amount of ABTS (2 mM) was used, and 0.25 mM NADH (final concentration) was added to the assay. Proteolysis was monitored as above by following the loss of absorbance at 340 nm due to the oxidation of NADH (e340 Å 6220 M01 cm01). Initial rates were measured directly from the time courses. NO2 –Phe Peptide Bond Cleavage Assay By use of a modified literature procedure (2), the hydrolysis of the peptide substrate (ATHQVYPhe(NO2)VRKA) by HIV protease tethered dimer was monitored by the decrease in absorbance at 300 nm (De Å 1360 M01 cm01). Kinetic constants were determined over a substrate range of 0.28–0.025 mM at an enzyme concentration of 1.2 mg/ml in 50 mM potassium acetate (pH 4.75) and 0.38 M KCl at 257C. Assays were conducted in 1 ml total volume in semi-micro cuvettes. Data were collected with a Shimadzu UVPC-2101 spectrophotometer.
FIG. 1. Relationship between free phenylalanine concentration and the maximum absorbance due to ABTS oxidation. The break in linearity above 25 mM phenylalanine is due to side reactions of the oxidized ABTS. Assays were conducted in (l) cuvettes of 1-cm pathlength or in (s) 96-well plates with an effective optical pathlength of 0.65 cm.
cm01 (Fig. 1). The break from linearity above 25 mM phenylalanine was due to a chemical reaction between oxidized ABTS and the a-keto acid produced by amino acid oxidase-catalyzed oxidation. The coupled assay produces similar results with leucine, methionine, tryptophan, tyrosine, and arginine (data not shown), with the exception that 10-fold more amino acid oxidase is required to produce maximal absorbance in less than 10 min with arginine. With the carboxypeptidase A peptide substrate, hippuryl-phenylalanine, the ABTS coupled assay produces a linear response from 3 to 25 mM with an effective absorptivity of 32 { 1 mM01 cm01 (Fig. 2). Figure 3 shows the actual time courses from which the data for Fig. 2 were taken. For all concentrations of hippuryl-phenylalanine employed, maximum absorbance was achieved within 6 min after addition of the coupling enzyme solution, and maximum absorbance was maintained for a minimum of 45 s.
Kinetic Analysis and Curve Fitting Enzyme kinetic data were fit by the method of nonlinear least squares to the equation for a rectangular hyperbola [v Å VmaxS/(Km / S)] by use of the Grafit program (Erithacus Software) obtained from Sigma Chemical Co.
RESULTS
ABTS Coupled Assay In either a cuvette or a 96-well plate the ABTS coupled assay provided a linear response to free phenylalanine from 5 mM up to a concentration of approximately 25 mM with an effective absorptivity of 35 { 2 mM01
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NADH Coupled Assay Although hippuryl-phenylalanine (N-benzoyl-glycylphenylalanine, Hip-Phe) is a good substrate for carboxypeptidase A [sp act 55 mmol/min/mg (as reported from Sigma Chemical Co.), Km Å 1.7 mM (3)], directly monitoring the rate of hydrolysis of Hip-Phe by its absorbance at 254 nm is not a particularly sensitive assay method (e Å 2500 M01 cm01; De for hydrolysis Å 368 M01 cm01). By use of the NADH coupled assay as described under Materials and Methods, a 1:1 stoichiometry for the amount of Hip-Phe to NADH oxidation was established (Table 1). When assays were conducted under these conditions at concentrations of carboxypeptidase A (0.6 mg/ml) that limited the rate of the reaction, a
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ENZYME-COUPLED ASSAYS FOR PROTEASES TABLE 1
Stoichiometry between NADH Oxidized and Concentration of Phenylalanine Phenylalanine (mM)
Expected DA (340 nm)
Observed DA (340 nm)
0.05 0.10
0.311 0.622
0.340 { 0.022 (n Å 2) 0.642 { 0.033 (n Å 4)
Note. The expected DA is based on the absorptivity of NADH (6220 cm01) assuming a 1:1 stoichiometry.
01
M
FIG. 2. Relationship between hippuryl-phenylalanine concentration and the maximum absorbance (derived from the time courses shown in Fig. 3). Assays were conducted in duplicate in cuvettes with a relative error of õ2%. 01
Vmax of 59 { 3 mmol min mM were determined.
mg
01
and a Km of 2.2 { 0.3
Endoprotease Assay (Pepsin) Despite the kinetic instability of the end point obtained in the ABTS couple in the absence of NADH, the greater intrinsic sensitivity of this assay has distinct advantages. For 22 min in a 50-ml volume at 257C, were incubated various amounts of pepsin (50 to 500 ng), 0.02 M sodium formate (pH 3.75), 0.45 M KCl, and 0.8 mM peptide substrate (Ala-His-Gln-Ala-Phe-Phe-ValArg-Lys-Ala-amide). At a concentration of 100 ng of pepsin, the change in absorbance indicated that 6% (10 mM) of the total substrate (0.16 mM final concentration) had been cleaved. The absorbance obtained in this assay was proportional to the amount of pepsin added
up to an absorbance of 0.8 (data not shown). These assays were repeated with 0.5 mg of pepsin and 3.7 mM to 4.5 mM peptide amide present. A Vmax of 23 { 1 mmol min01 mg01 and a Km of 0.08 { 0.01 mM were obtained. Endoprotease Assay (HIV Protease) For 3 min in a 200-ml total volume at 257C, 30 ng HIV protease tethered dimer was incubated with 0.017 to 1.1 mM peptide substrate (Ala-Thr-His-Gln-Val-TyrPhe(NO2)-Val-Arg-Lys-Ala) at pH 4.75 in 50 mM potassium acetate and 0.38 M KCl. A 50-ml aliquot of this preincubation assay solution was then transferred to 200 ml of the ABTS coupling enzyme solution. The maximum change in absorbance indicated that less than 10% of the total peptide substrate had been cleaved during the preincubation. A Vmax of 30.6 { 0.4 mmol min01 mg01 and a Km of 0.112 { 0.005 mM were obtained. For comparison with a standard method, the above assay was run again but monitored by use of the change in absorbance at 300 nm upon hydrolysis of the Tyr-Phe(NO2) bond as described under Materials and Methods. A Vmax of 27 { 3 mmol min01 mg01 and a Km of 0.11 { 0.03 mM were obtained. DISCUSSION
FIG. 3. Time course for color development after addition of a coupling solution (ABTS coupled assay) to various concentrations of hippuryl-phenylalanine.
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ABTS is an excellent substrate for horseradish peroxidase and has large extinctions when oxidized (e Å 36,000 and 12,000 M01 cm01 at 414 and 730 nm, respectively) (4); however, the oxidized dye reacts with the aketo acid produced in the oxidation of free amino acids by amino acid oxidase and does not give a kinetically stable end point. Although NADH (e340 nm Å 6220 M01 cm01) is not a substrate for the peroxidase, oxidized ABTS will rapidly oxidize NADH and act as a mediator to effectively couple NADH oxidation to hydrogen peroxide reduction. By use of this couple we were able to accurately determine the stoichiometry of the amino acid oxidase coupled reaction as one equivalent of NADH oxidized per phenylalanine produced (Table 1). Further, we were able to confirm that, in the absence of an NADH couple, ABTS oxidation is a reliable and quantitative estimate of the amount of phenylalanine produced (up to a total absorbancy at 414 nm of 0.8,
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Fig. 1), even though the color does not persist for an extended period of time after formation. Although it was possible to conduct the ABTS coupled assay in a continuous fashion with carboxypeptidase A (data not shown), only fixed-time assays were practical with pepsin and, to a lesser extent, the HIV protease due to proteolytic inactivation of amino acid oxidase. Conducting the assay in two stages, preincubation for endoproteolytic cleavage followed by addition of coupling enzymes (with concomitant change in pH and ionic strength), allows greater flexibility in the assay conditions used for a particular protease. Increasing the pH to 7.5 by addition of the coupling solution will stop the action of pepsin. One limitation of the assay described here is its sensitivity to reductants that can react with oxidized ABTS, such as a-keto acids and thiols. Use of catalytic amounts of ABTS and high concentrations of NADH (NADH chemical couple) substantially eliminates interference of the assay by reductants, but with loss of assay sensitivity. With the exception of aspartate, glutamate, arginine, lysine, proline, or hydroxyproline, bovine carboxypeptidase A will cleave an L-amino acid from the carboxy terminus of a peptide that has a free a-carboxyl group (5). If a peptide has an arginine or lysine at its carboxy terminus, then the commercially available porcine carboxypeptidase B can be used to cleave the peptide bond between this amino acid and the penultimate residue (6). Yeast carboxypeptidase Y can rapidly hydrolyze the amide bond of a carboxy-terminal glutamate, arginine, or proline or slowly hydrolyze that of aspartate, depending on the nature of the penultimate residue (glycine giving little or no rate of cleavage) (7). The porcine kidney aminopeptidase will cleave an L-amino acid from the amino terminus of a peptide with a specificity similar to carboxypeptidase A (8). By using a combination of these commercially available exopeptidases (carboxypeptidases A, B, and Y and aminopeptidase) virtually any endoproteolytic cleavage of a peptide blocked at its carboxyl and/or amino terminus could result in the liberation of free amino acids through the action of these exoproteases. If the free amino acid is leucine, methionine, phenylalanine, tryptophan, tyrosine, or arginine, then the endoproteolytic cleavage could effectively be coupled to the sensitive chromophore ABTS through the action of horseradish peroxidase as described herein. With the exoprotease carboxypeptidase A and the endoproteases pepsin and HIV-1 protease we have demonstrated the utility of a new coupled protease assay. For each of these proteases, the kinetic parameters obtained are in good agreement with more conventional protease assays (Table 2). Typical protease assays use substrates that have high intrinsic absorbances with small changes upon proteolysis, require time-consuming HPLC analysis, or require chromogenic or fluorogenic components that are expensive
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TABLE 2
Comparison of the Kinetic Parameters Determined by the Coupled Assays Described Herein with Other Methods or with Literature Values Kinetic parameters
Enzyme Carboxypeptidase A Pepsin HIV protease
Current method Vmaxa Kmb Vmaxa Kmb Vmaxa Kmb
59 2.2 23 0.08 30.6 0.112
{ { { { { {
3 0.3 1 0.01 0.4 0.005
Reported values or other methods 55c, 37e 3.0d, 1.75e 31f 0.005 f 27 { 3g 0.11 { 0.03g
a
The values for Vmax are reported as mmol/min/mg. The values for Km are reported in mM. c Value determined by Sigma Chemical Co. by use of the change in absorbance at 254 nm upon hydrolysis of hippuryl-phenylalanine. Assays were conducted at 257C in an unspecified buffer at pH 7.5. d Value determined in this study by use of the change in absorbance at 254 nm upon hydrolysis of hippuryl-phenylalanine. Assays were conducted at 257C in 0.025 M Trisrchloride buffer, pH 7.5, containing 0.5 M LiCl. e Value reported by Folk and Schirmer (3) by use of the change in absorbance at 254 nm upon hydrolysis of hippuryl-phenylalanine. Assays were conducted at 257C in 0.025 M Trisrchloride buffer, pH 7.5, containing 0.5 M NaCl. f Value reported by Agarwal and Rich (9) by use of the chromogenic peptide substrate Phe-Ala-Ala-Phe(NO2)-Phe-Val-Leu-OM4P (a different substrate than the one employed in the current method) monitoring the change in absorbance at 310 nm. Assays were conducted at 257C in 0.01 M sodium formate buffer at pH 3.5. g Value determined in this study by use of the chromogenic peptide substrate Ala-Thr-His-Gln-Val-Tyr-Phe(NO2)-Val-Arg-Lys-Ala monitoring the change in absorbance at 300 nm as reported by Cheng et al. (2). Assays were conducted at 257C in 50 mM potassium acetate buffer, pH 4.75, containing 0.38 M KCl. b
and/or time-consuming to synthesize. Where simple, sensitive, chromogenic substrates such as p-nitroanilide amides are amenable, a new assay method is not needed. However, if these substrates are not utilized by any new enzyme of interest, few alternatives exist. For the vast majority of these proteases the coupled enzyme assay described herein is most suitable. REFERENCES 1. Cheng, Y.-S. E., McGowen, M. H., Kettner, C. A., Schloss, J. V., Erickson-Viitanen, S., and Yin, F. H. (1990) Gene 87, 243–248. 2. Cheng, Y.-S. E., Yin, S.-H., Foundling, S., Blomstrom, D., and Kettner, C. A. (1990) Proc. Natl. Acad. Sci. USA 87, 9660–9664. 3. Folk, J. E., and Schirmer, E. W. (1963) J. Biol. Chem. 238, 3884– 3894. 4. Childs, R. E., and Bardsley, W. G. (1975) Biochem. J. 145, 93–103. 5. Petra, P. H. (1970) Methods Enzymol. 19, 460–503. 6. Folk, J. E. (1970) Methods Enzymol. 19, 504–521. 7. Hayashi, R. (1976) Methods Enzymol. 45B, 568–587. 8. Pfleiderer, G. (1970) Methods Enzymol. 19, 514–534. 9. Agarwal, N., and Rich, D. H. (1983) Anal. Biochem. 130, 158–165.
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241, 1–4 (1996)
0368
Enzyme-Coupled Assays for Proteases1 Brian E. Cathers and John V. Schloss2 Department of Medicinal Chemistry, 4070 Malott Hall, University of Kansas, Lawrence, Kansas 66045
Received November 30, 1995
MATERIALS AND METHODS We have developed a general strategy for assaying proteases that does not require the use of fluorogenic, chromogenic, or radiolabeled peptide substrates. The endoor exoproteolytic hydrolysis of simple peptides can be followed spectrophotometrically by coupling the proteolytic event via enzyme-catalyzed reactions to a chromogenic redox dye. The couple can be used directly to follow the action of carboxy or amino peptidases on peptide substrates or can be coupled by use of carboxy or amino peptidases to follow the action of endoproteases on peptide substrates that are blocked at the amino or carboxy terminus, respectively. Liberated amino acids are detected by use of amino acid oxidase, oxygen, horseradish peroxidase, and the redox dye 2,2*-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (e414 nm Å 36,000 M01 cm01). q 1996 Academic Press, Inc.
An assay strategy for proteases that employs simple, well-characterized, low-molecular-weight peptides composed of the 20 common amino acids would have great utility in characterizing the kinetic behavior of novel proteases. If such an assay were spectrophotometric-based and adaptable to a 96-well plate format, it would have the additional advantages of being quantitative, convenient, and useful for high-capacity screening of potential inhibitors. After exploring several potential enzyme-coupled assays for proteases, we have developed a protocol that meets the aforementioned criteria. Described below are the specifics of what, to the best of our knowledge, constitutes the first enzyme-coupled, spectrophotometric assay for proteases that employs simple nonchromophoric peptides. 1
The authors gratefully acknowledge financial support from the General Research Fund of the University of Kansas, the J. R. and Inez W. Jay Research Fund, the National Institutes of Health (GM07775), and the Burroughs Wellcome Co. We also thank Dr. Bruce Korant and Dr. Charles A. Kettner of the DuPont–Merck Pharmaceutical Co. for their generous gift of research materials. 2 To whom correspondence should be addressed. Fax: (913) 8645326. E-mail: [email protected].
Biochemicals L-Amino acid oxidase (Crotalus adamanteus EC 1.4.3.2), 2,2*-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),3 bovine carboxypeptidase A (EC 3.4.2.1), hippuryl-phenylalanine, horseradish peroxidase (EC 1.11.1.7), and porcine pepsin (EC 3.4.4.1) were purchased from Sigma Chemical Co. The tethered, single-polypeptide form of recombinant HIV protease, as prepared by the method described by Cheng et al. (1, 2), was a gift from Dr. Bruce Korant (DuPont– Merck Pharmaceuticals). The peptide substrate (ATHQVYPhe(NO2)VRKA) was a gift from Dr. Charles A. Kettner (DuPont–Merck Pharmaceuticals). The peptide substrate (AHQAFFVRKA-amide) was prepared by use of the DuPont RaMPS protocol with the Rapid Amide resin. Stock solutions of bovine carboxypeptidase A (1 mg/ml) were prepared by dissolving the aqueous suspension in 2.4 M LiCl/10 mM TrisrHCl (pH 7.5). Stocks of pepsin (5 mg/ml) were prepared by dissolving the lyophilized powder in sodium formate buffer (10 mM, pH 3.75). Horseradish peroxidase stocks (5–6 mg/ ml) and peptide substrate stocks (5–20 mM) were prepared as unbuffered aqueous solutions.
ABTS Coupled Assay The coupling enzymes, buffers, and reagents were employed at the following final concentrations: 0.1 M LiCl, 0.05 M TrisrHCl (pH 7.5), 2 mg/ml carboxypeptidase A, 70 mg/ml amino acid oxidase, 50 mg/ml horseradish peroxidase, and 0.8 mM ABTS. This coupling enzyme solution should be prepared at least 20 min before use and can be stored for up to 5 h at 257C. If carboxypeptidase A is omitted (to be added just prior to use), then the coupling solution is stable for up to 16 h at room temperature. Endoproteases were preincubated with peptide substrate at 257C for fixed periods of time in 20 to 50 mM 3 Abbreviation used: ABTS, 2,2*-azino-bis-(3-ethylbenzthiazoline6-sulfonic acid.
1
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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buffer (sodium formate, pH 3.75; potassium acetate, pH 4.75; MesrNaOH, pH 6.2; or TrisrHCl, pH 7.4) with various concentrations of KCl (0, 0.15, 0.38, or 0.45 M). Aliquots of the endoprotease assay solution were added to the coupling enzyme solution to a final volume of 1 ml in a cuvette or 0.25 ml in a 96-well plate. The increase in absorbance at 414 nm was monitored with a Shimadzu UVPC-2101 spectrophotometer or with a Molecular Devices THERMOmax microplate reader in kinetic mode using a 414-nm filter. Reaction rates were determined from the maximum absorbance obtained upon addition of the coupling enzyme solution. NADH Coupled Assay A modified version of the above assay that ‘‘chemically coupled’’ (nonenzymically) the oxidized ABTS to NADH was used initially for the assay of carboxypeptidase A. A catalytic amount of ABTS (2 mM) was used, and 0.25 mM NADH (final concentration) was added to the assay. Proteolysis was monitored as above by following the loss of absorbance at 340 nm due to the oxidation of NADH (e340 Å 6220 M01 cm01). Initial rates were measured directly from the time courses. NO2 –Phe Peptide Bond Cleavage Assay By use of a modified literature procedure (2), the hydrolysis of the peptide substrate (ATHQVYPhe(NO2)VRKA) by HIV protease tethered dimer was monitored by the decrease in absorbance at 300 nm (De Å 1360 M01 cm01). Kinetic constants were determined over a substrate range of 0.28–0.025 mM at an enzyme concentration of 1.2 mg/ml in 50 mM potassium acetate (pH 4.75) and 0.38 M KCl at 257C. Assays were conducted in 1 ml total volume in semi-micro cuvettes. Data were collected with a Shimadzu UVPC-2101 spectrophotometer.
FIG. 1. Relationship between free phenylalanine concentration and the maximum absorbance due to ABTS oxidation. The break in linearity above 25 mM phenylalanine is due to side reactions of the oxidized ABTS. Assays were conducted in (l) cuvettes of 1-cm pathlength or in (s) 96-well plates with an effective optical pathlength of 0.65 cm.
cm01 (Fig. 1). The break from linearity above 25 mM phenylalanine was due to a chemical reaction between oxidized ABTS and the a-keto acid produced by amino acid oxidase-catalyzed oxidation. The coupled assay produces similar results with leucine, methionine, tryptophan, tyrosine, and arginine (data not shown), with the exception that 10-fold more amino acid oxidase is required to produce maximal absorbance in less than 10 min with arginine. With the carboxypeptidase A peptide substrate, hippuryl-phenylalanine, the ABTS coupled assay produces a linear response from 3 to 25 mM with an effective absorptivity of 32 { 1 mM01 cm01 (Fig. 2). Figure 3 shows the actual time courses from which the data for Fig. 2 were taken. For all concentrations of hippuryl-phenylalanine employed, maximum absorbance was achieved within 6 min after addition of the coupling enzyme solution, and maximum absorbance was maintained for a minimum of 45 s.
Kinetic Analysis and Curve Fitting Enzyme kinetic data were fit by the method of nonlinear least squares to the equation for a rectangular hyperbola [v Å VmaxS/(Km / S)] by use of the Grafit program (Erithacus Software) obtained from Sigma Chemical Co.
RESULTS
ABTS Coupled Assay In either a cuvette or a 96-well plate the ABTS coupled assay provided a linear response to free phenylalanine from 5 mM up to a concentration of approximately 25 mM with an effective absorptivity of 35 { 2 mM01
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NADH Coupled Assay Although hippuryl-phenylalanine (N-benzoyl-glycylphenylalanine, Hip-Phe) is a good substrate for carboxypeptidase A [sp act 55 mmol/min/mg (as reported from Sigma Chemical Co.), Km Å 1.7 mM (3)], directly monitoring the rate of hydrolysis of Hip-Phe by its absorbance at 254 nm is not a particularly sensitive assay method (e Å 2500 M01 cm01; De for hydrolysis Å 368 M01 cm01). By use of the NADH coupled assay as described under Materials and Methods, a 1:1 stoichiometry for the amount of Hip-Phe to NADH oxidation was established (Table 1). When assays were conducted under these conditions at concentrations of carboxypeptidase A (0.6 mg/ml) that limited the rate of the reaction, a
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ENZYME-COUPLED ASSAYS FOR PROTEASES TABLE 1
Stoichiometry between NADH Oxidized and Concentration of Phenylalanine Phenylalanine (mM)
Expected DA (340 nm)
Observed DA (340 nm)
0.05 0.10
0.311 0.622
0.340 { 0.022 (n Å 2) 0.642 { 0.033 (n Å 4)
Note. The expected DA is based on the absorptivity of NADH (6220 cm01) assuming a 1:1 stoichiometry.
01
M
FIG. 2. Relationship between hippuryl-phenylalanine concentration and the maximum absorbance (derived from the time courses shown in Fig. 3). Assays were conducted in duplicate in cuvettes with a relative error of õ2%. 01
Vmax of 59 { 3 mmol min mM were determined.
mg
01
and a Km of 2.2 { 0.3
Endoprotease Assay (Pepsin) Despite the kinetic instability of the end point obtained in the ABTS couple in the absence of NADH, the greater intrinsic sensitivity of this assay has distinct advantages. For 22 min in a 50-ml volume at 257C, were incubated various amounts of pepsin (50 to 500 ng), 0.02 M sodium formate (pH 3.75), 0.45 M KCl, and 0.8 mM peptide substrate (Ala-His-Gln-Ala-Phe-Phe-ValArg-Lys-Ala-amide). At a concentration of 100 ng of pepsin, the change in absorbance indicated that 6% (10 mM) of the total substrate (0.16 mM final concentration) had been cleaved. The absorbance obtained in this assay was proportional to the amount of pepsin added
up to an absorbance of 0.8 (data not shown). These assays were repeated with 0.5 mg of pepsin and 3.7 mM to 4.5 mM peptide amide present. A Vmax of 23 { 1 mmol min01 mg01 and a Km of 0.08 { 0.01 mM were obtained. Endoprotease Assay (HIV Protease) For 3 min in a 200-ml total volume at 257C, 30 ng HIV protease tethered dimer was incubated with 0.017 to 1.1 mM peptide substrate (Ala-Thr-His-Gln-Val-TyrPhe(NO2)-Val-Arg-Lys-Ala) at pH 4.75 in 50 mM potassium acetate and 0.38 M KCl. A 50-ml aliquot of this preincubation assay solution was then transferred to 200 ml of the ABTS coupling enzyme solution. The maximum change in absorbance indicated that less than 10% of the total peptide substrate had been cleaved during the preincubation. A Vmax of 30.6 { 0.4 mmol min01 mg01 and a Km of 0.112 { 0.005 mM were obtained. For comparison with a standard method, the above assay was run again but monitored by use of the change in absorbance at 300 nm upon hydrolysis of the Tyr-Phe(NO2) bond as described under Materials and Methods. A Vmax of 27 { 3 mmol min01 mg01 and a Km of 0.11 { 0.03 mM were obtained. DISCUSSION
FIG. 3. Time course for color development after addition of a coupling solution (ABTS coupled assay) to various concentrations of hippuryl-phenylalanine.
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ABTS is an excellent substrate for horseradish peroxidase and has large extinctions when oxidized (e Å 36,000 and 12,000 M01 cm01 at 414 and 730 nm, respectively) (4); however, the oxidized dye reacts with the aketo acid produced in the oxidation of free amino acids by amino acid oxidase and does not give a kinetically stable end point. Although NADH (e340 nm Å 6220 M01 cm01) is not a substrate for the peroxidase, oxidized ABTS will rapidly oxidize NADH and act as a mediator to effectively couple NADH oxidation to hydrogen peroxide reduction. By use of this couple we were able to accurately determine the stoichiometry of the amino acid oxidase coupled reaction as one equivalent of NADH oxidized per phenylalanine produced (Table 1). Further, we were able to confirm that, in the absence of an NADH couple, ABTS oxidation is a reliable and quantitative estimate of the amount of phenylalanine produced (up to a total absorbancy at 414 nm of 0.8,
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Fig. 1), even though the color does not persist for an extended period of time after formation. Although it was possible to conduct the ABTS coupled assay in a continuous fashion with carboxypeptidase A (data not shown), only fixed-time assays were practical with pepsin and, to a lesser extent, the HIV protease due to proteolytic inactivation of amino acid oxidase. Conducting the assay in two stages, preincubation for endoproteolytic cleavage followed by addition of coupling enzymes (with concomitant change in pH and ionic strength), allows greater flexibility in the assay conditions used for a particular protease. Increasing the pH to 7.5 by addition of the coupling solution will stop the action of pepsin. One limitation of the assay described here is its sensitivity to reductants that can react with oxidized ABTS, such as a-keto acids and thiols. Use of catalytic amounts of ABTS and high concentrations of NADH (NADH chemical couple) substantially eliminates interference of the assay by reductants, but with loss of assay sensitivity. With the exception of aspartate, glutamate, arginine, lysine, proline, or hydroxyproline, bovine carboxypeptidase A will cleave an L-amino acid from the carboxy terminus of a peptide that has a free a-carboxyl group (5). If a peptide has an arginine or lysine at its carboxy terminus, then the commercially available porcine carboxypeptidase B can be used to cleave the peptide bond between this amino acid and the penultimate residue (6). Yeast carboxypeptidase Y can rapidly hydrolyze the amide bond of a carboxy-terminal glutamate, arginine, or proline or slowly hydrolyze that of aspartate, depending on the nature of the penultimate residue (glycine giving little or no rate of cleavage) (7). The porcine kidney aminopeptidase will cleave an L-amino acid from the amino terminus of a peptide with a specificity similar to carboxypeptidase A (8). By using a combination of these commercially available exopeptidases (carboxypeptidases A, B, and Y and aminopeptidase) virtually any endoproteolytic cleavage of a peptide blocked at its carboxyl and/or amino terminus could result in the liberation of free amino acids through the action of these exoproteases. If the free amino acid is leucine, methionine, phenylalanine, tryptophan, tyrosine, or arginine, then the endoproteolytic cleavage could effectively be coupled to the sensitive chromophore ABTS through the action of horseradish peroxidase as described herein. With the exoprotease carboxypeptidase A and the endoproteases pepsin and HIV-1 protease we have demonstrated the utility of a new coupled protease assay. For each of these proteases, the kinetic parameters obtained are in good agreement with more conventional protease assays (Table 2). Typical protease assays use substrates that have high intrinsic absorbances with small changes upon proteolysis, require time-consuming HPLC analysis, or require chromogenic or fluorogenic components that are expensive
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TABLE 2
Comparison of the Kinetic Parameters Determined by the Coupled Assays Described Herein with Other Methods or with Literature Values Kinetic parameters
Enzyme Carboxypeptidase A Pepsin HIV protease
Current method Vmaxa Kmb Vmaxa Kmb Vmaxa Kmb
59 2.2 23 0.08 30.6 0.112
{ { { { { {
3 0.3 1 0.01 0.4 0.005
Reported values or other methods 55c, 37e 3.0d, 1.75e 31f 0.005 f 27 { 3g 0.11 { 0.03g
a
The values for Vmax are reported as mmol/min/mg. The values for Km are reported in mM. c Value determined by Sigma Chemical Co. by use of the change in absorbance at 254 nm upon hydrolysis of hippuryl-phenylalanine. Assays were conducted at 257C in an unspecified buffer at pH 7.5. d Value determined in this study by use of the change in absorbance at 254 nm upon hydrolysis of hippuryl-phenylalanine. Assays were conducted at 257C in 0.025 M Trisrchloride buffer, pH 7.5, containing 0.5 M LiCl. e Value reported by Folk and Schirmer (3) by use of the change in absorbance at 254 nm upon hydrolysis of hippuryl-phenylalanine. Assays were conducted at 257C in 0.025 M Trisrchloride buffer, pH 7.5, containing 0.5 M NaCl. f Value reported by Agarwal and Rich (9) by use of the chromogenic peptide substrate Phe-Ala-Ala-Phe(NO2)-Phe-Val-Leu-OM4P (a different substrate than the one employed in the current method) monitoring the change in absorbance at 310 nm. Assays were conducted at 257C in 0.01 M sodium formate buffer at pH 3.5. g Value determined in this study by use of the chromogenic peptide substrate Ala-Thr-His-Gln-Val-Tyr-Phe(NO2)-Val-Arg-Lys-Ala monitoring the change in absorbance at 300 nm as reported by Cheng et al. (2). Assays were conducted at 257C in 50 mM potassium acetate buffer, pH 4.75, containing 0.38 M KCl. b
and/or time-consuming to synthesize. Where simple, sensitive, chromogenic substrates such as p-nitroanilide amides are amenable, a new assay method is not needed. However, if these substrates are not utilized by any new enzyme of interest, few alternatives exist. For the vast majority of these proteases the coupled enzyme assay described herein is most suitable. REFERENCES 1. Cheng, Y.-S. E., McGowen, M. H., Kettner, C. A., Schloss, J. V., Erickson-Viitanen, S., and Yin, F. H. (1990) Gene 87, 243–248. 2. Cheng, Y.-S. E., Yin, S.-H., Foundling, S., Blomstrom, D., and Kettner, C. A. (1990) Proc. Natl. Acad. Sci. USA 87, 9660–9664. 3. Folk, J. E., and Schirmer, E. W. (1963) J. Biol. Chem. 238, 3884– 3894. 4. Childs, R. E., and Bardsley, W. G. (1975) Biochem. J. 145, 93–103. 5. Petra, P. H. (1970) Methods Enzymol. 19, 460–503. 6. Folk, J. E. (1970) Methods Enzymol. 19, 504–521. 7. Hayashi, R. (1976) Methods Enzymol. 45B, 568–587. 8. Pfleiderer, G. (1970) Methods Enzymol. 19, 514–534. 9. Agarwal, N., and Rich, D. H. (1983) Anal. Biochem. 130, 158–165.
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