Protease and Protease Inhibitor Assays Using Biotinylated Casein Coated on a Solid Phase

Analytical Biochemistry 268, 151–156 (1999) Article ID abio.1998.3053, available online at http://www.idealibrary.com on

Protease and Protease Inhibitor Assays Using Biotinylated Casein Coated on a Solid Phase 1 Zhibo Gan,* Ronald R. Marquardt,* ,2 and Hao Xiao† *Department of Animal Science, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada; and †ImmuneChem Pharmaceuticals Inc., Burnaby, BC, V5J 3M6 Canada

Received October 20, 1998

A new type of solid-phase assay for proteases and protease inhibitors has been developed using biotinylated casein. The assay involves coating of titer plate wells with biotinylated casein, hydrolysis of this substrate with a protease such as trypsin, reaction of the biotin from the unhydrolyzed substrate with an alkaline phosphatase–streptavidin complex, and finally quantification of the amount of casein remaining on the plate using alkaline phosphatase activity as the indicator. The activity of the bound indicator enzyme is oppositely related to the protease activity of the sample. In addition, the assay can be modified for quantitating the corresponding amount of protease inhibitor in the sample. The assay is simple, sensitive, accurate, inexpensive, and amenable to automation. © 1999 Academic Press Key Words: protease; inhibitor; biotinylated casein; solid-phase assay.

The biochemical and physiological functions of protease in part have been well established (1, 2). Recent evidence, however, suggests that proteases also play an important role in the growth and proliferation of pathogenic bacteria and enhancing pathogenesis of severe disease (3). Protease, as a virulence factor, has stimulated the search for potent disease-curing drugs that can inhibit the activity of protease, an example of which is the HIV protease inhibitor (4). During screening for promising drug candidates, hundreds of thousands of compounds need to be assayed. Generally, up to 100,000 samples must be analyzed in each high-throughput screening (HTS) 3 assay in order to identify about 10 to 1

A patent is pending for the method described in this paper. To whom correspondence should be addressed. Fax: 204-4747628. E-mail: [email protected]. 3 Abbreviations used: HTS, high-throughput screening; pNPP, p-nitrophenyl phosphate disodium; BAPNA, Na-benzoyl- -arginine p-nitronilide; PBS, phosphate-buffered saline; PBST, PBS plus 0.05% Tween 20. 2

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0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

100 leads that are biologically active (5). Obviously, the availability of a simple, sensitive, and efficient method for assaying protease activity would facilitate the testing of a large number of samples using HTS. Several endeavors have been made in recent years to meet this need. Fluorescence polarization (FP) technology (6), FITC 25–BSA (7), BODIPY dye-labeled casein (8), and HPLC (9) have been used for the assay of protease activity. These procedures can fulfill the requirements of HTS only to varying degrees. Therefore, there is a need for improved alternate types of assays. The objective of the current study was to develop a simple assay for proteases and protease inhibitors. In this assay biotinylated casein that has been immobilized onto a microtiter plate is incubated with the protease of interest and the amount of unhydrolyzed casein– biotin complex is quantitated enzymatically following binding of an alkaline phosphatase–streptavidin conjugate to the residual biotin. The color produced by the activity of the bound alkaline phosphatase is indirectly related to the activity of the protease in the media. The use of a second indicator enzyme in the assay greatly amplifies the signal. The use of a solid-state titer plate assay coupled with the use of a second enzyme yields an assay that is simple, sensitive, and efficient. The method is amenable to automation and therefore to high throughput. MATERIALS AND METHODS

Materials Biotinylated casein was from Norzyme Inc. (Winnipeg, Canada). The molar ratio of biotin to casein was 3:1. Tween 20, p-nitrophenyl phosphate disodium (pNPP), trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), thermolysin (EC 3.4.24.3), collagenase (EC 3.4.24.3), pepsin (EC 3.4.23.1), elastase (EC 3.4.21.37), protease XIII (Aspergillus saitoi) (EC 3.4.23.18), thrombin (EC 3.4.21.5), aprotinin, a 2-macroglobulin, iodoacetic acid, pepstatin A, Na-benzoyl- -arginine p-nitronilide (BAPNA), and L

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ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma Chemical Co. (St. Louis, MO), dimethyl sulfoxide (DMSO) from J. T. Baker Chemical Co. (Phillipsburg, NJ), and microplates (Falcon 3911) from Becton–Dickinson and Co. (Oxnard, CA). Citrate (0.1 M)–phosphate (0.2 M) buffers (10) were used for papain (pH 6.2), protease XIII (pH 2.8), pepsin (pH 2.2), and elastase (pH 6.5) and phosphate (0.2 M) buffers (6) for trypsin (pH 7.5), thermolysin (pH 7.5), and collagenase (pH 7.1). Methods Sensitivity of the assay as affected by the concentration of biotinylated casein coated onto the surface of the microplate well. The microplates except for the first column of eight wells were coated using biotinylated casein in PBS at 0.05, 0.1, and 1 mg/100 ml/well and incubated in a humid atmosphere for 2 h at 37°C. The plates were washed three times with 10 mM PBST (PBS with 0.05% Tween 20). Assay procedure for protease activity. Different concentrations of the enzyme solution as indicated in Figs. 1 and 2 (100 ml) were prepared with the appropriate buffer (see Materials and Table 1 for further details). The enzymes were added in triplicate to the wells of a plate from the 2nd column to the 11th column. The 1st column is a blank and contained only buffer but no coated substrate, while the 12th column is the positive control and contained only buffer and coated substrate. The plate was incubated in a humid atmosphere at room temperature for 30 min followed by washing three times using PBST to terminate the protease reaction. A streptavidin–alkaline phosphatase solution (100 ml/well, 1:1000 dilution in bicarbonate buffer, pH 9.5) was added to all wells in the plate. The plate was incubated at room temperature for 30 min. The reaction was stopped by emptying and washing the plate five times with PBST. The pNPP substrate (100 ml/ well, 1 mg/ml in 10% diethanolamine buffer, pH 9.8) was added to each well of the plate followed by incubation at room temperature for 25 min. This time was sufficient to give absorbance values of between 2.0 and 2.5 in the control samples with no protease. The absorbance was then read at 405 nm using a microplate reader (Bio-Rad Laboratories, Mississauga, Ontario, Canada; Model 450). The unknown activity of samples to be assayed was determined from the standard curve (Figs. 1 and 2). Assay procedure for concentration of inhibitor. Buffer (100 ml) was added to each well in the 1st column (as the blank, it contained no coated substrate) and the 2nd column (as the positive control, it contained coated substrate). Additional buffer (50 ml) was added to each well in the 3rd column (as the negative control) and all remaining wells except those in the 4th column. The inhibitor solution (100 ml) was added in triplicate to wells in

the 4th column (see Table 2 for examples of target enzymes that were used for the different classes of inhibitors). In addition, the ability of an unusual inhibitor, a 2-macroglobulin, to inhibit trypsin was also tested in pH 7.2 phosphate buffer (10 mM). The activity of trypsin was 1 U/100 ml/well while the concentration of a 2-macroglobulin was from 0.0001 to 0.01 nM/100 ml/well. Fifty microliters of inhibitor solution was taken from wells in the 4th column and added to wells in the 5th column and mixed well. This step was repeated until the 12th column after which the samples were mixed well and 50 ml from the 12th column was discarded. This provided different concentrations of serially diluted inhibitor. A fixed amount of enzyme solution (50 ml) was added (see Table 2) to each well of the plate except for those in the 1st and 2nd columns. The plate was incubated at room temperature for 30 min followed by washing three times using PBST to terminate the protease inhibitor reaction. A streptavidin–alkaline phosphatase solution (100 ml/well, 1:1000 dilution in bicarbonate buffer, pH 9.5) was added to all wells in the plate. The plate was incubated at room temperature for 30 min. The reaction was stopped by emptying and washing the plate five times with PBST. The pNPP substrate (100 ml/well, 1 mg/ml in 10% diethanolamine buffer, pH 9.8) was added to each well of the plate followed by incubation at room temperature for 25 min. The absorbance was then read at 405 nm using a microplate reader (Bio-Rad Laboratories; Model 450). Percentage of inhibition was calculated according to the equation Inhibition activity % 5

ODi 2 ODn 3 100, ODp 2 ODn

where ODi is the OD value of wells with enzyme and inhibitor, ODn is the OD value of negative control (3rd column), and ODp is the OD value of positive control (2nd column). A standard curve can be constructed using these data (Fig. 3). The concentration of inhibitor in unknown samples was determined from the standard curve. The BAPNA method for determining enzyme activity. The BAPNA is commonly used as a substrate for assay of trypsin. Tryptic hydrolysis of the synthetic substrate releases r-nitroaniline which is yellow and can be measured colorimetrically. The assay procedure was essentially as describe by Erlanger et al. (11). Briefly, five concentrations of trypsin for standard curve and five samples with unknown activity of trypsin were added to a reaction vessel containing 5 mM BAPNA and incubated at room temperature for 15 min. The absorbance was then read at 405 nm. The unknown activities of samples were determined from the standard curve. RESULTS

Effect of coating on the sensitivity of assay. Biotinylated casein at concentrations of 0.05, 0.1, and 1 mg/100

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FIG. 1. Effect of coating concentration on the sensitivity of the assay. Values represent means of triplicate analysis. CV was less than 5% for triplicate analysis. See Materials and Methods for further details.

ml/well was used to demonstrate the effect of coating concentration on the sensitivity of the assay (Fig. 1). Considerable improvements in the sensitivity of the assay can be achieved by the coating of smaller amounts of labeled casein onto the surface of the well. This reduction (0.05 mg/100 ml/well), however, resulted in a less steep curve (Fig. 1) and a decrease in the rate of color development by the avidin–alkaline phosphatase conjugate (data not shown). Higher coating concentration (1 mg/100 ml/ well) resulted in a reduced degree of sensitivity. Therefore, a concentration of 0.1 mg/100 ml/well for coating was used for further research. Protease activity assay. The biotinylated a-casein that was bound onto the surface of the wells of a microtiter plate can be used as a substrate for all of the proteases tested in this study (Table 1). These enzymes can be grouped into four classes of proteases including those which have activated serine, cysteine, aspartate,

and metal ion (12). Typical curves for the hydrolysis of the labeled casein by enzymes from each of the different classes of proteases (trypsin, papain, protease XIII, and collagenase) were illustrated in Figs. 2a–2d. The sensitivity of the assay varied depending on the enzyme used (Table 1). The enzyme with the greatest sensitivity was thermolysin (0.0000025 U/100 ml/well), while pepsin had the lowest sensitivity (16 U/100 ml/ well). Thrombin, an enzyme with high substrate specificity (13), did not hydrolyze the substrate. Protease inhibitor assay. Aprotinin, iodoacetic acid, pepstatin A, and EDTA were selected as models for inhibiting the four classes of proteases (Table 2). Inhibition of the target enzymes (trypsin, papain, pepsin, and collagenase) by the inhibitors demonstrated that the biotinylated casein method can be used to quantitate the amount of protease inhibitor in a sample (Figs. 3a–3d, Table 2). It also can be used to assay the amount of a 2 -macroglobulin. The concentrations of a 2-macroglobulin to 10, 50, and 90% inhibition were 0.0006, 0.0022, and 0.0038 nM/100 ml. Since the I 50 or K i values among inhibitors are different (14), the sensitivity of the inhibitor assay using the same target enzyme is different among inhibitors. Comparison of the biotin– casein method with the BAPNA method. The biotin– casein method as developed in this paper and the BAPNA method (11) which is commonly used as an esterase assay for trypsin were compared. Five concentrations of trypsin were assayed in triplicate using the two methods and these values were compared using the t test (Corel Quattro Pro software, Ottawa, Ontario, Canada). The means 6 SD were 4.72 6 2.38 and 4.82 6 1.61, respectively, for the BAPNA and biotin– casein assays. t test results when the two assays were compared showed that utu 5 0.077 , t 0.05. These data therefore demonstrated that there were no differences (P . 0.05) between the two

TABLE 1

Hydrolysis of Biotinylated Casein by Different Classes of Proteases Enzyme Serine protease Trypsin (EC 3.4.21.4) Elastase (EC 3.4.21.3) Cysteine protease Papain (EC 3.4.22.2) Aspartic protease Pepsin (EC 3.4.23.1) Protease XIII (EC 3.4.23.18) Metalloprotease Thermolysin (EC 3.4.24.27) Collagenase (EC 3.4.24.3) a b

Activity a (U/mg)

Sensitivity b (U/100 ml)

Range for assay (U/100 ml)

Buffer

0.002 0.00006

0.001–1 0.00001–0.1

10 mM phosphate, pH 7.2 10 mM phosphate, pH 7.8

15

0.001

0.0001–0.01

10 mM phosphate, pH 6.5

3,900 0.9

16 0.00018

1–10,000 0.000001–1

Citrate–phosphate, pH 2.2 Citrate–phosphate, pH 2.8

0.000001–1 0.1–10

10 mM phosphate, pH 6.5 50 mM Tris –HCL, pH 7.5

14,900 5

40 2,510

0.0000025 0.22

Units of activity (U/mg) was defined by supplier. Sensitivity was defined as the activity of enzyme required to hydrolyze 10% of the substrate.

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FIG. 2. Hydrolysis of biotinylated casein by proteases including trypsin, papain, protease XIII, and collagenase. Values represent means of triplicate analysis. CV was less than 5% for triplicate analysis. See Materials and Methods for further details.

sets of data when the enzyme was assayed using either biotin– casein or BAPNA. The lower detection limit for the biotin– casein assay for trypsin was 0.001 U/100 ml (Table 1) while that of BAPNA was 2 U/100 ml under the experimental conditions. The detection limits (sensitivity) were considered to be the concentration of enzymes that could be detected when the absorbance change was 10% of maximum absorbance change. Er-

langer et al. (11), who developed the BAPNA method, obtained similar results. DISCUSSION

The solid-phase biotinylated casein method as reported in this paper presents a new approach for the assay of proteases and protease inhibitors. The binding

TABLE 2

Inhibition of Proteases by Inhibitors a Target enzyme b

Inhibitor

50% inhibition (nM/100 ml)

Trypsin Papain Pepsin Collagenase

Aprotinin Iodoacetic acid Pepstatin A EDTA

0.0028 13.4 6.8 2429

Range for assay (nM/100 ml) 0.001 –0.1 1 –100 3 –100 500 –10000

Buffer 10 10 10 50

mM mM mM mM

phosphate, pH 7.2 phosphate, pH 6.5 phosphate, pH 6.5 Tris–HCL, pH 7.5

a The molecular weights of the different enzymes were trypsin, 23,800 Da; papain, 20,200 Da; pepsin, 35,000 Da (18); and collagenase (not available). The molecular weights of the different inhibitors were aprotinin, 6500 Da; iodoacetic acid, 185.9 Da; pepstatin A, 685.9 Da; and EDTA, 292.2 Da (Sigma Catalog 1998). b The activities or amounts of enzyme used in the inhibitor assay were 1 U or 0.00028 nM/100 ml for trypsin, 0.02 U or 0.066 nM/100 ml for papain, 1950 U or 14.3 nM/100 ml for pepsin, and 10 U/100 ml for collagenase.

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FIG. 3. Inhibition of protease activity by inhibitors. The enzymes used and the corresponding inhibitors were trypsin inhibited by aprotinin, pepsin inhibited by pepstatin A, collagenase inhibited by EDTA, and papain inhibited by iodoacetic acid. Values represent means of triplicate analysis. CV was less than 5% for triplicate analysis. See Materials and Methods for further details.

of the substrate to a solid phase greatly facilitates subsequent steps in the assay such as washing to remove hydrolyzed products, unreacted reagents, or impurities in the assay extract, while use of a labeled substrate allows for the ready analysis of the amount of substrate that remains. Binding of biotin, a relatively small molecule, to the substrate reduces the degree of steric interference that may occur with larger indicator molecules and also provides considerable flexibility in the type of indicator that can be used, as the biotin will react with any indicator provided it is conjugated to avidin, a protein that has a high affinity for biotin (15). Also, use of an enzyme such as alkaline phosphatase as the indicator molecule, rather than a colored molecule, allows for a markedly amplified signal since each phosphatase can generate many colored molecules. The adaptation of the entire process to a microplate format and the use of an ELISA reader that is coupled to a computer to measure absorbency and calculate results greatly facilitate the analysis of many samples in a relatively short period of time. In addition, the assay requires only small amounts of reagents, has high sensitivity and accuracy, and can be completed in a relatively short period of time. The assay, as a result, can

be automated and is suitable for analysis of a large number of samples. The biotinylated casein method has overcome drawbacks of radioactive hazard of the radioactive assay (16) and time-consuming manipulations (e.g., trichloroacetic precipitation, centrifugation, and heating) (17) and expensive equipment requirements (6) associated with other assays. It has a very high sensitivity, fulfills the essential requirements for the assay of most proteases, and has the capability of testing activity over a wide range. Also, the nature of the assay, as indicated under Results, allows considerable flexibility in its design and therefore in its corresponding sensitivity. Results have demonstrated that the sensitivity of the assay can be increased by decreasing the amount of substrate coated onto the wells of the titer plate (Fig. 1) and increasing hydrolysis time (data not shown). These changes can produce dramatic improvements in sensitivity, but in some cases corresponding increases in the time required to produce the desired absorbency changes are required. This later problem, however, can be solved by use of different indicator enzymes such as horseradish peroxidase or by use of an avidin complex that has multiple units of the indicator bound to it and/or by use of chemi-

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luminescent substrates. A reduced concentration of coating substrate may result in nonspecific binding of the protease to the surface of the microplate well. This effect could possibly be corrected by use of a nonprotein blocking agent such as dextran. It should therefore be possible not only to develop more sensitive protease assays but also to have an assay that can be completed in a relatively short period of time. The assay, however, requires more steps than the BAPNA assay which uses a synthetic substrate. The disadvantage of the BAPNA assay is that it is only specific for enzymes such as trypsin that have both esterase and protease activities. The BAPNA assay therefore cannot be used to assay other proteases and it is less sensitive than the assay reported in this study. The class of protease in the sample can be readily identified by comparing the results with those obtained with reference inhibitors, each of which would inhibit a different class of protease. In addition, the assay can be modified for determination of other inhibitors such as a 2macroglobulin, an unusual and specific protease inhibitor. Overall, this paper reports on the development of a new type of assay for proteases and protease inhibitors that is sensitive, accurate, simple, rapid, and readily adapted to the specificity of the sample to be determined. The assay is also inexpensive to carry out, can utilize equipment that is present in most laboratories, and can be readily automated. ACKNOWLEDGMENTS Financial support for Zhibo Gan from a strategic grant from the National Sciences and Engineering Research Council of Canada and from the University of Manitoba is appreciated.

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