ANALYTICAL
BIOCHEMISTRY
A Microassay James
195,129-131
(1991)
for Proteolytic
Activity
J. Plantner
Lorand V. Johnson Laboratory for Eye Research,Department of Surgery, Division of Ophthalmology, CaseWesternReserveUniver&, Cl&eland, Ohio 44106
Received
November
30,199O
A quantitative procedure for measuring proteolytic activity, utilizing azoalbumin as substrate, has been developed for use in microtiter plates. An enzyme-linked immunosorbent assay reader is used to measure absorbance. The procedure is sensitive, as well as being both rapid and economical. It is particularly convenient for measuring large numbers of samples, such as fractions o 1991 Academic PWE, IIN. from column chromatography.
A number of methods are available for measuring the activity of proteolytic enzymes (1). Synthetic substrates, radioactively labeled materials, and natural proteins with chromophoric or fluorogenic groups attached have been utilized, along with the measurement of amino acids released during digestion. While studying the neutral metalloproteinases present in bovine interphotoreceptor matrix (IPM)’ (2), a microproteinase assay was developed, utilizing the inexpensive, commercially available substrate, azoalbumin (3). While this method is both sensitive and economical, its main strength lies in the ease of processing of multiple samples, e.g., monitoring column fractions, etc. Since the color of the final chromophore is developed in microtiter plates, it may simply be viewed to establish the presence of proteolytic activity, or measured in an ELISA reader for quantitation. Other procedures for the measurement of proteolytic activity in microtiter plates have been described (4-5). However, they have utilized fluorogenic peptides as substrates, many of which are not commercially available or are relatively expensive. Furthermore, while the products of proteolytic activity in those procedures can be qualitatively viewed by use of a uv lamp, quantitative
1 Abbreviations used: IPM, interphotoreceptor matrix; ELISA, zyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl oride; RPE, retinal pigment epithelium. 0003-2697/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
enflu-
measurement may be a bit more difficult, fluorometer. METHODS
AND
requiring a
MATERIALS
Interphotoreceptor natrix. Interphotoreceptor matrix was prepared as described previously (2), by a modification of the method of Adler and Severin (6). Briefly, cornea, lens, aqueous and vitreous humor, and retina were removed from fresh bovine eyes. Cold buffer (Dulbecco’s phosphate-buffered saline, containing 10 mM EDTA, 3 mM PMSF, 5 pg/ml benzamidine, 3 pg/ml pepstatin A, and 2 pglml leupeptin) was added to the eyecup and pipetted against the apical surface of the retinal pigment epithelium (RPE) a number of times to produce the RPE-IPM. This was then centrifuged at 48,000g for 30 min to remove the remaining debris, and the supernatant fluid was concentrated to the equivalent of about 5 eyes/ml by ultrafiltration using Amicon (Danvers, MA) YM-10 membranes and stored at -20°C. Gel filtration. RPE-IPM was applied to a 1.6 X 95 cm column of Sephacryl S-500, superfine (Pharmacia, Uppsala), equilibrated with 0.05 M Na phosphate, pH 7.5,0.1 M NaCl. The material was eluted at a flow rate of 10 ml/h. Fractions were extensively dialyzed against water before analysis. All column procedures were at 4°C. Incubations were h&roassay for proteolytic activity. performed at 37°C in 1.5-ml microcentrifuge tubes. Each incubation contained azoalbumin, 0.4 mg, and 0.1 M MOPS0 buffer, pH 7.0, along with enzyme solution and/or additives in a final volume of 0.25 ml. Incubations were stopped by the addition of 0.04 ml of 50% trichloroacetic acid. The samples were allowed to stand at 4’C for 1 h, and the resulting precipitates removed by centrifugation at 500g for 15 min. Aliquots of the supernatant fluids (0.2 ml) were then added to the wells of a microtiter plate with sufficient force to produce mixing with the 0.015 ml of 10 N NaOH already present in each 129
Inc.
reserved.
130
JAMES
20
10
Time
J. PLANTNER
30
(min)
FIG. 1.
Proteolytic activity vs time. Papain (final concentration of 0.025 mg/ml) was incubated at 37°C for the times indicated as described under Materials and Methods. Azoalbumin was present at a concentration of 1.6 mg/ml. The points are the average of three closely spaced replicates.
well. Alkalimzation is necessary to enhance sensitivity, since in acid the released azo dye absorbs much more weakly. Absorbance was automatically measured using a Dynatech (Chantilly, Virginia) MR 5000 ELISA reader with a 450-nm test filter and a 630-nm reference filter. Appropriate blanks were prepared. Materials. The following materials were obtained from Sigma (St. Louis): azoalbumin; MOPSO; trypsin, type III from bovine pancreas, 13,900 BAEE units/mg; papain, type III, 244 BAEE units/mg; thermolysin (proteinase type X), 60 pmol tyrosine released/min/mg; collagenase, type VII from Clostridium histolyticum, 1400 units/mg; pepstatin A; PMSF; leupeptin; and benzamidine. All other materials were of reagent grade.
be obtained in the alkalinized supernatant solutions while still maintaining an excess of the substrate. This assay was applied to incubations measuring trypsin, papain, and thermolysin, representing the serine, cysteine, and metallo classes of proteinase, respectively (7). The substrate was cleaved by papain in a manner which was linear in terms of both time, for at least 30 min (Fig. l), and protein (Fig. 2). Replicate determinations were closely spaced, having an average SE of 0.017 for measurements like those in Figs. 1 and 2. Similar results were obtained with trypsin and thermolysin (data not shown). The assay thus provides a quantitative measure of the relative activity of a single proteinase under different conditions and allows a comparison of the relative activity of various proteinasesunder the same conditions. Based on the activity of the added enzyme in terms of BAEE units, papain was relatively much more reactive than trypsin but similar to thermolysin (data not shown). Collagenase produced no cleavage, even after prolonged incubations with 75 units of enzyme. Effect of inhibitors. Most standard proteinase inhibitors do not interfere with this procedure, thus it can be utilized when determining the class of an unknown proteinase. As seen in Table 1, all of the proteinase inhibitors displayed the expected results. Many divalent cations, in concentrations higher than 1 mM, however, did interfere with the assay, either by precipitating the released chromophore or by producing interfering colors. Ca’+, Zn2+, and Mg2+ were exceptions, and could be used at concentration up to 10 mM without interference. A number of microbial inhibitors, such as sodium azide, toluene, thimerosal, penicillin/streptomycin/fungizone, etc., did not interfere with the assay. This assay can also be used to measure the levels of
RESULTS AND DISCUSSION Microassay. The major impetus for developing this procedure was to obtain an assay which was simple, inexpensive, and rapid, since our studies (2) necessitated measuring many fractions separated by column chromatography. Azoalbumin (3) was chosen as substrate since it is readily cleaved by most proteinases and it is quite inexpensive. The original procedure which utilized this reagent required fairly large volumes and the time-consuming reading of individual fractions in cuvettes. Use of a spectrophotometer with a sipper attachment for the absorbance measurements of the alkalinized supernatants improved upon the speed of the original assay. However, by transferring the supernatants to microtiter plates containing alkali, and using an ELISA reader to obtain the absorbance, much greater savings of time were realized. The smaller volumes used in this assay also enhanced sensitivity and saved reagents. A concentration of 1.6 mg/ml was chosen for the substrate, azoalbumin, so that a final absorbance of about 2 could
Final
Concentration
(mg/ml)
FIG. 2. Proteolytic activity vs enzyme concentration. Incubations were performed at 37°C for 2 h as described under Materials and Methods. Tubes contained papain at 0.01 to 0.06 mg/ml final concentration and azoalbumin at 1.6 mg/ml. The points are the average of three closely spaced replicates.
MICRO
PROTEINASE
tissue inhibitors by incubating a constant amount of a standard proteinase with varying amounts of tissue extract or aliquots of fractions from a separation. The 450-nm absorbance resulting from the proteinase will be decreased where inhibitor is present. Depending on the class of proteinase used in the incubation, different types of inhibitors can be detected specifically. This procedure has been used to detect the metalloproteinase inhibitors of IPM (2). Interphotoreceptor matrix metalloproteinuses. The IPM is the extracellular matrix which lies between the RPE and the neural retina (8). This assay has been used to measure the presence of proteolytic activity in RPEIPM. As seen in Fig. 3, proteolytic activity was present through much of the fractionation range of Sephacryl S-500, suggesting the existence of a number of different proteinases in this extracellular matrix. All of this activity has been shown to belong to the metalloproteinase class, since it was inhibited by phenanthroline (data not shown) (7). In summary, a simple, inexpensive, rapid assay, capable of measuring a broad range of proteolytic activities, has been developed. The assay is especially useful where many samples are involved. It has been used to measure the activity of a number of standard proteinases and matrix metalloproteinases from bovine IPM. The assay uses the commercially available substrate, azoalbumin
TABLE
1
Inhibitor EDTA (pH 7.0) l,lO-Phenanthroline’ Aprotinin PMSF Leupeptin E-64C Na Iodoacetate pCMB
50 10 40 10 300 10 30 10
mM mM
pglml mM
@g/ml aglml mM mM
Trypsin NP NI 2 pglml 5mM
20 pglml NI NI 5mM
Papain UD,) NI
0.05mM NI NI 10 fig/ml 1 rglml 0.1 mM 0.1 mM
1.0 A .z 0.8
.iy
0.6
0 p
9-
0.4
0.2
a $ 5 h 0 A z 6 2
0.0
Fraction
Number
(3 ml)
FIG. 3. Fractionation of proteolytic activity by gel filtration of RPE-IPM. RPE-IPM (10 ml) was fractionated on Sephacryl S-500. Fractions (x3 ml) were collected and diluted where necessary to measure uv absorbance, the difference of A2,6nm - Asznrn (Cl) is plotted (9). Aliquots (0.1 ml) of individual fractions (16-63) were analyzed for proteinase activity (A) as described under Materials and Methods. Relative activity has been normalized. Incubations were at 37°C for 42 h. Toluene (3%) was included in the incubations to prevent microbial contamination.
(3). The released chromophore is measured in microtiter plates using an ELISA reader. It is likely that this procedure could also be adapted for the use of azocasein or casein yellow for proteinases preferring casein as a substrate. ACKNOWLEDGMENTS
Effect of Proteinase Inhibitors Maximum dose tested
131
ASSAY
Thermolysin 0.01 mM 1mM NI NI NI NI 10 mM NI
Note. Trypsin, papain, and thermolysin” were incubated with various potential inhibitors and azoalbumin (1.6 mg/ml) as described under Materials and Methods. The concentration of inhibitor necessary to produce 50% inhibition of activity (ID,) was determined. ’ Final concentrations were as follows: trypsin, 50 wg/ml; papain, 20 pg/ml; thermolysin, 5 pg/ml. The concentration of proteinase was chosen such that similar absorbance at 450 nm was obtained from each in uninhibited incubations. * Activity not inhibited at least 50% even at the maximum dose tested. ’ Dissolved in ethanol.
Appreciation is expressed to Dr. Min Jiang for the expert technical assistance provided. This work was supported by Public Health Service Research Grant EY 06571 from the National Eye Institute and the Ohio Lions’ Eye Research Foundation.
REFERENCES 1. Sarath, G., de la Motte, R. S., and Wagner, F. W. (1989) in Proteolytic Enzymes: A Practical Approach (Beynon, R. J., and Bond, J. S., Eds.), pp. 25-55, IRL Press, Oxford. 2. Plantner, J. J. (1990) Invest. Ophthulmol. Vis. Sci. Suppl. 31, 72. 3. Tomarelli, R. M., Charney, J., and Harding, M. L. (1949) J. Lab. Clin. Med. 34, 426-433. 4. Dresden, M. H., Rotmans, J. P., Deelder, A. M., Koper, G., and Ploem, J. S. (1982) Anal. Biochem. 126,170-173. 5. Irvine, G. B., Ennis, M., and Williams, C. H. (1990) Anal. Biothem. 186.304-307. 6. Adler, A. J., and Severin, K. M. (1981) Exp. Eye Res. 32,755-769. 7. Bond, J. S., and Butler, P. E. (1987) Anna Rev. Biochem. 66, 333-364. 8. Rohlich, P. (1970) Exp. Eye Res. 10,80-86. 9. Waddell,
W. J. (1956)
J. Lab.
Clin. Med.
48.311-314.
BIOCHEMISTRY
A Microassay James
195,129-131
(1991)
for Proteolytic
Activity
J. Plantner
Lorand V. Johnson Laboratory for Eye Research,Department of Surgery, Division of Ophthalmology, CaseWesternReserveUniver&, Cl&eland, Ohio 44106
Received
November
30,199O
A quantitative procedure for measuring proteolytic activity, utilizing azoalbumin as substrate, has been developed for use in microtiter plates. An enzyme-linked immunosorbent assay reader is used to measure absorbance. The procedure is sensitive, as well as being both rapid and economical. It is particularly convenient for measuring large numbers of samples, such as fractions o 1991 Academic PWE, IIN. from column chromatography.
A number of methods are available for measuring the activity of proteolytic enzymes (1). Synthetic substrates, radioactively labeled materials, and natural proteins with chromophoric or fluorogenic groups attached have been utilized, along with the measurement of amino acids released during digestion. While studying the neutral metalloproteinases present in bovine interphotoreceptor matrix (IPM)’ (2), a microproteinase assay was developed, utilizing the inexpensive, commercially available substrate, azoalbumin (3). While this method is both sensitive and economical, its main strength lies in the ease of processing of multiple samples, e.g., monitoring column fractions, etc. Since the color of the final chromophore is developed in microtiter plates, it may simply be viewed to establish the presence of proteolytic activity, or measured in an ELISA reader for quantitation. Other procedures for the measurement of proteolytic activity in microtiter plates have been described (4-5). However, they have utilized fluorogenic peptides as substrates, many of which are not commercially available or are relatively expensive. Furthermore, while the products of proteolytic activity in those procedures can be qualitatively viewed by use of a uv lamp, quantitative
1 Abbreviations used: IPM, interphotoreceptor matrix; ELISA, zyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl oride; RPE, retinal pigment epithelium. 0003-2697/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
enflu-
measurement may be a bit more difficult, fluorometer. METHODS
AND
requiring a
MATERIALS
Interphotoreceptor natrix. Interphotoreceptor matrix was prepared as described previously (2), by a modification of the method of Adler and Severin (6). Briefly, cornea, lens, aqueous and vitreous humor, and retina were removed from fresh bovine eyes. Cold buffer (Dulbecco’s phosphate-buffered saline, containing 10 mM EDTA, 3 mM PMSF, 5 pg/ml benzamidine, 3 pg/ml pepstatin A, and 2 pglml leupeptin) was added to the eyecup and pipetted against the apical surface of the retinal pigment epithelium (RPE) a number of times to produce the RPE-IPM. This was then centrifuged at 48,000g for 30 min to remove the remaining debris, and the supernatant fluid was concentrated to the equivalent of about 5 eyes/ml by ultrafiltration using Amicon (Danvers, MA) YM-10 membranes and stored at -20°C. Gel filtration. RPE-IPM was applied to a 1.6 X 95 cm column of Sephacryl S-500, superfine (Pharmacia, Uppsala), equilibrated with 0.05 M Na phosphate, pH 7.5,0.1 M NaCl. The material was eluted at a flow rate of 10 ml/h. Fractions were extensively dialyzed against water before analysis. All column procedures were at 4°C. Incubations were h&roassay for proteolytic activity. performed at 37°C in 1.5-ml microcentrifuge tubes. Each incubation contained azoalbumin, 0.4 mg, and 0.1 M MOPS0 buffer, pH 7.0, along with enzyme solution and/or additives in a final volume of 0.25 ml. Incubations were stopped by the addition of 0.04 ml of 50% trichloroacetic acid. The samples were allowed to stand at 4’C for 1 h, and the resulting precipitates removed by centrifugation at 500g for 15 min. Aliquots of the supernatant fluids (0.2 ml) were then added to the wells of a microtiter plate with sufficient force to produce mixing with the 0.015 ml of 10 N NaOH already present in each 129
Inc.
reserved.
130
JAMES
20
10
Time
J. PLANTNER
30
(min)
FIG. 1.
Proteolytic activity vs time. Papain (final concentration of 0.025 mg/ml) was incubated at 37°C for the times indicated as described under Materials and Methods. Azoalbumin was present at a concentration of 1.6 mg/ml. The points are the average of three closely spaced replicates.
well. Alkalimzation is necessary to enhance sensitivity, since in acid the released azo dye absorbs much more weakly. Absorbance was automatically measured using a Dynatech (Chantilly, Virginia) MR 5000 ELISA reader with a 450-nm test filter and a 630-nm reference filter. Appropriate blanks were prepared. Materials. The following materials were obtained from Sigma (St. Louis): azoalbumin; MOPSO; trypsin, type III from bovine pancreas, 13,900 BAEE units/mg; papain, type III, 244 BAEE units/mg; thermolysin (proteinase type X), 60 pmol tyrosine released/min/mg; collagenase, type VII from Clostridium histolyticum, 1400 units/mg; pepstatin A; PMSF; leupeptin; and benzamidine. All other materials were of reagent grade.
be obtained in the alkalinized supernatant solutions while still maintaining an excess of the substrate. This assay was applied to incubations measuring trypsin, papain, and thermolysin, representing the serine, cysteine, and metallo classes of proteinase, respectively (7). The substrate was cleaved by papain in a manner which was linear in terms of both time, for at least 30 min (Fig. l), and protein (Fig. 2). Replicate determinations were closely spaced, having an average SE of 0.017 for measurements like those in Figs. 1 and 2. Similar results were obtained with trypsin and thermolysin (data not shown). The assay thus provides a quantitative measure of the relative activity of a single proteinase under different conditions and allows a comparison of the relative activity of various proteinasesunder the same conditions. Based on the activity of the added enzyme in terms of BAEE units, papain was relatively much more reactive than trypsin but similar to thermolysin (data not shown). Collagenase produced no cleavage, even after prolonged incubations with 75 units of enzyme. Effect of inhibitors. Most standard proteinase inhibitors do not interfere with this procedure, thus it can be utilized when determining the class of an unknown proteinase. As seen in Table 1, all of the proteinase inhibitors displayed the expected results. Many divalent cations, in concentrations higher than 1 mM, however, did interfere with the assay, either by precipitating the released chromophore or by producing interfering colors. Ca’+, Zn2+, and Mg2+ were exceptions, and could be used at concentration up to 10 mM without interference. A number of microbial inhibitors, such as sodium azide, toluene, thimerosal, penicillin/streptomycin/fungizone, etc., did not interfere with the assay. This assay can also be used to measure the levels of
RESULTS AND DISCUSSION Microassay. The major impetus for developing this procedure was to obtain an assay which was simple, inexpensive, and rapid, since our studies (2) necessitated measuring many fractions separated by column chromatography. Azoalbumin (3) was chosen as substrate since it is readily cleaved by most proteinases and it is quite inexpensive. The original procedure which utilized this reagent required fairly large volumes and the time-consuming reading of individual fractions in cuvettes. Use of a spectrophotometer with a sipper attachment for the absorbance measurements of the alkalinized supernatants improved upon the speed of the original assay. However, by transferring the supernatants to microtiter plates containing alkali, and using an ELISA reader to obtain the absorbance, much greater savings of time were realized. The smaller volumes used in this assay also enhanced sensitivity and saved reagents. A concentration of 1.6 mg/ml was chosen for the substrate, azoalbumin, so that a final absorbance of about 2 could
Final
Concentration
(mg/ml)
FIG. 2. Proteolytic activity vs enzyme concentration. Incubations were performed at 37°C for 2 h as described under Materials and Methods. Tubes contained papain at 0.01 to 0.06 mg/ml final concentration and azoalbumin at 1.6 mg/ml. The points are the average of three closely spaced replicates.
MICRO
PROTEINASE
tissue inhibitors by incubating a constant amount of a standard proteinase with varying amounts of tissue extract or aliquots of fractions from a separation. The 450-nm absorbance resulting from the proteinase will be decreased where inhibitor is present. Depending on the class of proteinase used in the incubation, different types of inhibitors can be detected specifically. This procedure has been used to detect the metalloproteinase inhibitors of IPM (2). Interphotoreceptor matrix metalloproteinuses. The IPM is the extracellular matrix which lies between the RPE and the neural retina (8). This assay has been used to measure the presence of proteolytic activity in RPEIPM. As seen in Fig. 3, proteolytic activity was present through much of the fractionation range of Sephacryl S-500, suggesting the existence of a number of different proteinases in this extracellular matrix. All of this activity has been shown to belong to the metalloproteinase class, since it was inhibited by phenanthroline (data not shown) (7). In summary, a simple, inexpensive, rapid assay, capable of measuring a broad range of proteolytic activities, has been developed. The assay is especially useful where many samples are involved. It has been used to measure the activity of a number of standard proteinases and matrix metalloproteinases from bovine IPM. The assay uses the commercially available substrate, azoalbumin
TABLE
1
Inhibitor EDTA (pH 7.0) l,lO-Phenanthroline’ Aprotinin PMSF Leupeptin E-64C Na Iodoacetate pCMB
50 10 40 10 300 10 30 10
mM mM
pglml mM
@g/ml aglml mM mM
Trypsin NP NI 2 pglml 5mM
20 pglml NI NI 5mM
Papain UD,) NI
0.05mM NI NI 10 fig/ml 1 rglml 0.1 mM 0.1 mM
1.0 A .z 0.8
.iy
0.6
0 p
9-
0.4
0.2
a $ 5 h 0 A z 6 2
0.0
Fraction
Number
(3 ml)
FIG. 3. Fractionation of proteolytic activity by gel filtration of RPE-IPM. RPE-IPM (10 ml) was fractionated on Sephacryl S-500. Fractions (x3 ml) were collected and diluted where necessary to measure uv absorbance, the difference of A2,6nm - Asznrn (Cl) is plotted (9). Aliquots (0.1 ml) of individual fractions (16-63) were analyzed for proteinase activity (A) as described under Materials and Methods. Relative activity has been normalized. Incubations were at 37°C for 42 h. Toluene (3%) was included in the incubations to prevent microbial contamination.
(3). The released chromophore is measured in microtiter plates using an ELISA reader. It is likely that this procedure could also be adapted for the use of azocasein or casein yellow for proteinases preferring casein as a substrate. ACKNOWLEDGMENTS
Effect of Proteinase Inhibitors Maximum dose tested
131
ASSAY
Thermolysin 0.01 mM 1mM NI NI NI NI 10 mM NI
Note. Trypsin, papain, and thermolysin” were incubated with various potential inhibitors and azoalbumin (1.6 mg/ml) as described under Materials and Methods. The concentration of inhibitor necessary to produce 50% inhibition of activity (ID,) was determined. ’ Final concentrations were as follows: trypsin, 50 wg/ml; papain, 20 pg/ml; thermolysin, 5 pg/ml. The concentration of proteinase was chosen such that similar absorbance at 450 nm was obtained from each in uninhibited incubations. * Activity not inhibited at least 50% even at the maximum dose tested. ’ Dissolved in ethanol.
Appreciation is expressed to Dr. Min Jiang for the expert technical assistance provided. This work was supported by Public Health Service Research Grant EY 06571 from the National Eye Institute and the Ohio Lions’ Eye Research Foundation.
REFERENCES 1. Sarath, G., de la Motte, R. S., and Wagner, F. W. (1989) in Proteolytic Enzymes: A Practical Approach (Beynon, R. J., and Bond, J. S., Eds.), pp. 25-55, IRL Press, Oxford. 2. Plantner, J. J. (1990) Invest. Ophthulmol. Vis. Sci. Suppl. 31, 72. 3. Tomarelli, R. M., Charney, J., and Harding, M. L. (1949) J. Lab. Clin. Med. 34, 426-433. 4. Dresden, M. H., Rotmans, J. P., Deelder, A. M., Koper, G., and Ploem, J. S. (1982) Anal. Biochem. 126,170-173. 5. Irvine, G. B., Ennis, M., and Williams, C. H. (1990) Anal. Biothem. 186.304-307. 6. Adler, A. J., and Severin, K. M. (1981) Exp. Eye Res. 32,755-769. 7. Bond, J. S., and Butler, P. E. (1987) Anna Rev. Biochem. 66, 333-364. 8. Rohlich, P. (1970) Exp. Eye Res. 10,80-86. 9. Waddell,
W. J. (1956)
J. Lab.
Clin. Med.
48.311-314.
