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A sensitive assay for proteolytic activity using fluorescein-labeled gelatin coupled to Sepharose 4B as substrate
ANALYTICAL
BIOCHEMISTRY
152, 39-41 (1986)
A Sensitive Assay for Proteolytic Activity Using Fluorescein-Labeled Gelatin Coupled to Sepharose 46 as Substrate URSULA
TISLJAR
AND HANS-WERNER
DENKER
Abteilung Anatomie, RWTH, Aachen, West Germany Received June 4, 1985 A method for the determination of proteolytic activity is described which uses fluoresceinlabeled gelatin coupled to Sepharose 4B as substrate. The assay is simple and sensitive allowing detection of one nanogram of trypsin and is found suitable for the measurement of gelatinolytic activity in tissue samples. 0 1986 Academic Ress, Inc. KEY WORDS: proteinase assay;immobilized fluorescein-labeled gelatin. MATERIALS
A variety of methods for the quantitative measurement of proteinase activity has been developed. All suffer from some disadvantages: The use of chromogenic low-molecular-weight substrates is a very convenient way of measuring proteinase activity (1,2), but for some proteinases low-molecular-weight substrates have not been found (3). This approach is also not applicable when the peptide bond cleaved by a proteinase is unknown. In these cases it is operationally more reasonable to use labeled proteins as substrates (for a review see (4)). Dye-labeled proteins may be used, although such tests are not very sensitive (4-6). Tests with 3H-, 14C- or ‘%labeled substrates are sensitive but handicapped by the restrictions for working with radioactivity (4,7-9). Assays with enzyme-labeled proteins are very sensitive but need a second enzyme reaction and are therefore time consuming (4,10,11). Methods which are based on the determination of liberated amino groups cannot be used with crude tissue homogenates (2,4,12). In this report we describe a simple, sensitive, and inexpensive assay for the measurement of gelatinolytic activity which uses fluoresceinlabeled gelatin-Sepharose 4B as substrate. The test is well adapted to the measurement of proteinase activity in tissue homogenates.
AND
METHODS
Fluorescein isothiocyanate (No. 2 1590), trypsin (from bovine pancreas, twice crystalized, No. 37260), and elastase (from porcine pancreas, twice crystalized, No. 20930) were purchased from Serva Fine Biochemicals, Heidelberg. Gelatin (No. 4070) was obtained from Merck, Darmstadt; cyanobromide-activated Sepharose 4B from Pharmacia Fine Chemicals, Uppsala; and elastatinal (No. 2904 12) from Protein Research Foundation, Japan. Aprotinin (Trasylol) was a gift of Bayer AG, Leverkusen. For the labeling of gelatin with fluorescein (FITC),’ 250 mg gelatin was dissolved in 10 ml 0.1 M Na-phosphate buffer, pH 9.5. Five milliliters fluorescein isothiocyanate, 1 mg/ml, dissolved in the same buffer was added. The pH was checked continuously and if necessary adjusted to pH 9.5 during the 3 h of incubation at room temperature. The reaction was stopped by adding 1 M acetic acid to adjust the pH to 7.2. The labeled protein was dialyzed against distilled water for 48 h and afterwards lyophilized. Coupling of the FITC-labeled gelatin to Sepharose 4B. The coupling was done according ’ Abbreviation used: FITC, fluorescein isothiocyanate. 39
0003-2697186
$3.00
Copyright 0 1986 by Academic Pra, Inc. All right.5 of reproduction in any form reserved.
40
TISLJAR
AND DENKER
to Cuatrecasas ( 13) which is also the procedure recommended by the manufacturer. The standard procedure was done with 30 mg FITC-labeled gelatin and 1 g cyanobromideactivated Sepharose 4B. After the coupling, the free binding sites were blocked with glycine. The gel was washed alternatively with 80 ml 0.1 M Na-acetate buffer, pH 4, and 0.1 M NaHC03 buffer, pH 8.3, each containing 0.5 M NaCl five times, followed by one wash with 10 ml 0.05 M Tris-HCl buffer, pH 8.0, and suspension of the gel in 5 ml of the same TrisHCl buffer. Proteinase assay. The assay mixture consisted of 20 ~1 of FITC-labeled gelatin-Sepharose suspension, representing 112 pg gelatin substrate, 1O-200 ~1 enzyme sample, and 0.05 M Tris-HCl buffer, pH 8.0, to a final volume of 600 ~1. The samples were incubated in 1.5-ml Eppendorf microcentrifuge tubes at 37°C with continuous overhead shaking (30 cycles/min). In the standard procedure, the reaction was stopped after 60 min by chilling on ice. The tubes were centrifuged at 4°C. The fluorescence of the supernatant was measured in an Aminco SPF-500 spectrofluorometer with an exitation wavelength set at 480 nm and emission wavelength at 5 18 nm. Application of the methodfor tissue homogenates. Small pieces of pregnant rabbit endometrium (Days 6-7) were homogenized in 10 parts (w/v) of 0.05 M Tris-HCl buffer, pH 8.0, in a Potter-Elvehjem glass-teflon homogenizer. The homogenate was centrifuged at 1OOOgfor 10 min at 4°C. Proteinase activity was determined in lo-p1 aliquots of the supernatant as described above. Blank determinations of the tissue homogenate were carried out in the same way, except that buffer was added instead of FITC-labeled gelatinSepharose. Othercontrolsincludeddetermination of spontaneous hydrolysis of substrate and quenching of fluorescence by higher protein concentrations. The protein concentration of tissue samples was determined according to Lowry et al. ( 14) with bovine serum albumin as standard.
RESULTS AND DISCUSSION
The gelatin used in this study was labeled with 3 pg FITC/mg gelatin. Gel filtration on Sephadex G-25 showed that the labeled protein was free of unbound FITC. When coupled to cyanobromide-activated Sepharose 4B, 28 mg of the FITC-labeled gelatin were bound by 1 g dry Sepharose 4B. The immobilized FITClabeled gelatin was stable in buffer containing 0.02% Na azide at 4°C for at least 2 months. It is advisable to wash the immobilized substrate again before use, if stored for more than 1 week. As shown in Fig. 1, 1 ng of trypsin can be detected. An incubation time up to 60 min seems suitable for the assay with trypsin, since hydrolysis is linear during this time period (Fig. 2). The hydrolysis of FITC-labeled gelatin by trypsin was inhibited by aprotinin. The interassay coefficient of variation was 8.4% for values of 10 ng trypsin (n = 10). The FITC-labeled gelatin is also hydrolyzed by elastase. The detection limit is 10 ng for this proteinase. The cleavage is inhibited by elastatinal. It is possible to assay gelatinolytic activity in rabbit endometrium with the described test. Endometrial homogenate ( 10 ~1) corresponding to 100 pg protein hydrolyzed about the
FIG. 1. Hydrolysis of FITC-labeled gelatin as a function of trypsin concentration: 20 ~1 of FITC-labeled gelatinSepharose suspension, 0- 100 ng trypsin, and 0.05 M TrisHCI buffer, pH 8.0, to a final volume of 600 pl were incubated at 37°C with continuous shaking. After 60 min the tubes were chilled on ice and centrifuged, and the fluorescence of the supematant was measured without background correction in a spectrotluorometer (excitation wavelength 480 nm, emission wavelength 5 18 nm).
PROTEINASE
ASSAY USING IMMOBILIZED
FLUORESCEIN-GELATIN
41
ACKNOWLEDGMENTS This investigation was supported by Deutsche Forschungsgemeinschafi Grant De 18 l/9-5. The authors thank Prof. Dr. Greiling, Abteilung Klinische Chemie und Pathobiochemie, RWTH Aachen, for the opportunity to use his spectrofluorometer. or 0
20
LO
80
100
120
TIME6FIMIN~
2. Time course of digestion of FITC-labeled gelatin: 20 ~1 of FITC-labeled gelatin-Sephrose suspension and 10 ng trypsin in 580 1~10.05 M Tris-HCl buffer, pH 8.0, were incubated at 37°C with continuous shaking. After incubation time between 0 and 120 min, tubes were chilled on ice and centrifuged and the fluorescence of the supernatant was measured without background correction in a spectrofluorometer (excitation wavelength 480 nm, emission wavelength 5 18 nm).
REFERENCES
FIG.
same amount of FITC-labeled gelatin as 1 ng trypsin. The new assay is very simple, fast, and sufficiently reproducible. It has the advantage that no precipitation steps (2,4,7,15) or second enzyme reactions (4,10,11) are necessary. In comparison to a test with fluorescamine-labeled protein, the FITC-labeled substrate is remarkably stable and can be kept on stock rather then preparing the labeled substrate daily as necessary in the previous method ( 16). The stability of the substrate is comparable to a test with furanone-labeled substrates ( 17). Furthermore, if the described standard procedure is followed the tissue homogenate does not interfere with the detection of the fluorescence as in methods employing fluorescamine ( 18). Although no radioactive substrate is used, the test is as sensitive as a test with [3H]gelatin (7). It has proved useful for the detection of gelatinolytic activity in tissue samples. Furthermore, adaptation of the same test principle for the study of hydrolysis of other protein substrates labeled and immobilized in the same way should be possible.
1. Hemker, H. C. (ed.) (1983) in Handbook of Synthetic Substrates, pp. 27-47, Martinus Nijhoff, Boston. 2. McGillivray, D. H., Burnett, D., Afford, S. C., and Stockley, R. A. (1981) C/in. Chim Acta 111,289294.
Harris, E. D., and Cartwright, E. C. ( 1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J.. ed.), pp. 249-284, North-Holland, Amsterdam. 4. Wunderwald, P. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 258-270. Verlag Chemie, Weinheim. 5. Cheng, Y. S. E., and Aronson, A. I. (1977) Arch. Mi3.
crobiol.
115, 61-66.
6. Nelson, W. L., Ciaccio, E. I., and Hess, G. P. ( 196 1) Anal. Biochem.
2, 39-44.
7. Sunada, H., and Nagai, Y. (1980) J. Biochem. 87, 1765-1771. 8. Jensen, H. S., (1984) Biochem. J. 218,645-648. 9. Varani, J., Johnson, K., and Kaplan, J. (1980) Anal. Biochem.
107, 377-384.
10. Taylor, M. D., and Andrews, A. T. (1983) FEBSLett. 157,200-204.
11. Saunders, G. C., Svitra, Z., and Martinez, A. (1982) Anal. Biochem. 126, 122-l 30. 12. Sugiura, M., Ishikawa, M., Sasaki, M., Hirano, K., Ito, Y., and Awazu, S. (1979) Anal. Biochem. 97, 1l-16. 13. Cuatrecasas, P., Wilchek, M., and Anfinsen, C. B. (1968) Proc. Natl. Acad. Sci. USA 61, 636-643. 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (195 1) J. Biol. Chem. 193.265-275. 15. Twining, S. S. (1984) Anal. Biochem. 143, 30-34. 16. Ichihara, Y., Sogawa, K., and Takahashi, K. (1982) J. Biochem. 92, l-15. 17. Wiesner, R., and Troll, W. (1982) Anal. Biochem. 121, 290-294.
18. Evans, C. H., and Ridella, J. D. ( 1984) Anal. B&hem. 142,41
l-420.
BIOCHEMISTRY
152, 39-41 (1986)
A Sensitive Assay for Proteolytic Activity Using Fluorescein-Labeled Gelatin Coupled to Sepharose 46 as Substrate URSULA
TISLJAR
AND HANS-WERNER
DENKER
Abteilung Anatomie, RWTH, Aachen, West Germany Received June 4, 1985 A method for the determination of proteolytic activity is described which uses fluoresceinlabeled gelatin coupled to Sepharose 4B as substrate. The assay is simple and sensitive allowing detection of one nanogram of trypsin and is found suitable for the measurement of gelatinolytic activity in tissue samples. 0 1986 Academic Ress, Inc. KEY WORDS: proteinase assay;immobilized fluorescein-labeled gelatin. MATERIALS
A variety of methods for the quantitative measurement of proteinase activity has been developed. All suffer from some disadvantages: The use of chromogenic low-molecular-weight substrates is a very convenient way of measuring proteinase activity (1,2), but for some proteinases low-molecular-weight substrates have not been found (3). This approach is also not applicable when the peptide bond cleaved by a proteinase is unknown. In these cases it is operationally more reasonable to use labeled proteins as substrates (for a review see (4)). Dye-labeled proteins may be used, although such tests are not very sensitive (4-6). Tests with 3H-, 14C- or ‘%labeled substrates are sensitive but handicapped by the restrictions for working with radioactivity (4,7-9). Assays with enzyme-labeled proteins are very sensitive but need a second enzyme reaction and are therefore time consuming (4,10,11). Methods which are based on the determination of liberated amino groups cannot be used with crude tissue homogenates (2,4,12). In this report we describe a simple, sensitive, and inexpensive assay for the measurement of gelatinolytic activity which uses fluoresceinlabeled gelatin-Sepharose 4B as substrate. The test is well adapted to the measurement of proteinase activity in tissue homogenates.
AND
METHODS
Fluorescein isothiocyanate (No. 2 1590), trypsin (from bovine pancreas, twice crystalized, No. 37260), and elastase (from porcine pancreas, twice crystalized, No. 20930) were purchased from Serva Fine Biochemicals, Heidelberg. Gelatin (No. 4070) was obtained from Merck, Darmstadt; cyanobromide-activated Sepharose 4B from Pharmacia Fine Chemicals, Uppsala; and elastatinal (No. 2904 12) from Protein Research Foundation, Japan. Aprotinin (Trasylol) was a gift of Bayer AG, Leverkusen. For the labeling of gelatin with fluorescein (FITC),’ 250 mg gelatin was dissolved in 10 ml 0.1 M Na-phosphate buffer, pH 9.5. Five milliliters fluorescein isothiocyanate, 1 mg/ml, dissolved in the same buffer was added. The pH was checked continuously and if necessary adjusted to pH 9.5 during the 3 h of incubation at room temperature. The reaction was stopped by adding 1 M acetic acid to adjust the pH to 7.2. The labeled protein was dialyzed against distilled water for 48 h and afterwards lyophilized. Coupling of the FITC-labeled gelatin to Sepharose 4B. The coupling was done according ’ Abbreviation used: FITC, fluorescein isothiocyanate. 39
0003-2697186
$3.00
Copyright 0 1986 by Academic Pra, Inc. All right.5 of reproduction in any form reserved.
40
TISLJAR
AND DENKER
to Cuatrecasas ( 13) which is also the procedure recommended by the manufacturer. The standard procedure was done with 30 mg FITC-labeled gelatin and 1 g cyanobromideactivated Sepharose 4B. After the coupling, the free binding sites were blocked with glycine. The gel was washed alternatively with 80 ml 0.1 M Na-acetate buffer, pH 4, and 0.1 M NaHC03 buffer, pH 8.3, each containing 0.5 M NaCl five times, followed by one wash with 10 ml 0.05 M Tris-HCl buffer, pH 8.0, and suspension of the gel in 5 ml of the same TrisHCl buffer. Proteinase assay. The assay mixture consisted of 20 ~1 of FITC-labeled gelatin-Sepharose suspension, representing 112 pg gelatin substrate, 1O-200 ~1 enzyme sample, and 0.05 M Tris-HCl buffer, pH 8.0, to a final volume of 600 ~1. The samples were incubated in 1.5-ml Eppendorf microcentrifuge tubes at 37°C with continuous overhead shaking (30 cycles/min). In the standard procedure, the reaction was stopped after 60 min by chilling on ice. The tubes were centrifuged at 4°C. The fluorescence of the supernatant was measured in an Aminco SPF-500 spectrofluorometer with an exitation wavelength set at 480 nm and emission wavelength at 5 18 nm. Application of the methodfor tissue homogenates. Small pieces of pregnant rabbit endometrium (Days 6-7) were homogenized in 10 parts (w/v) of 0.05 M Tris-HCl buffer, pH 8.0, in a Potter-Elvehjem glass-teflon homogenizer. The homogenate was centrifuged at 1OOOgfor 10 min at 4°C. Proteinase activity was determined in lo-p1 aliquots of the supernatant as described above. Blank determinations of the tissue homogenate were carried out in the same way, except that buffer was added instead of FITC-labeled gelatinSepharose. Othercontrolsincludeddetermination of spontaneous hydrolysis of substrate and quenching of fluorescence by higher protein concentrations. The protein concentration of tissue samples was determined according to Lowry et al. ( 14) with bovine serum albumin as standard.
RESULTS AND DISCUSSION
The gelatin used in this study was labeled with 3 pg FITC/mg gelatin. Gel filtration on Sephadex G-25 showed that the labeled protein was free of unbound FITC. When coupled to cyanobromide-activated Sepharose 4B, 28 mg of the FITC-labeled gelatin were bound by 1 g dry Sepharose 4B. The immobilized FITClabeled gelatin was stable in buffer containing 0.02% Na azide at 4°C for at least 2 months. It is advisable to wash the immobilized substrate again before use, if stored for more than 1 week. As shown in Fig. 1, 1 ng of trypsin can be detected. An incubation time up to 60 min seems suitable for the assay with trypsin, since hydrolysis is linear during this time period (Fig. 2). The hydrolysis of FITC-labeled gelatin by trypsin was inhibited by aprotinin. The interassay coefficient of variation was 8.4% for values of 10 ng trypsin (n = 10). The FITC-labeled gelatin is also hydrolyzed by elastase. The detection limit is 10 ng for this proteinase. The cleavage is inhibited by elastatinal. It is possible to assay gelatinolytic activity in rabbit endometrium with the described test. Endometrial homogenate ( 10 ~1) corresponding to 100 pg protein hydrolyzed about the
FIG. 1. Hydrolysis of FITC-labeled gelatin as a function of trypsin concentration: 20 ~1 of FITC-labeled gelatinSepharose suspension, 0- 100 ng trypsin, and 0.05 M TrisHCI buffer, pH 8.0, to a final volume of 600 pl were incubated at 37°C with continuous shaking. After 60 min the tubes were chilled on ice and centrifuged, and the fluorescence of the supematant was measured without background correction in a spectrotluorometer (excitation wavelength 480 nm, emission wavelength 5 18 nm).
PROTEINASE
ASSAY USING IMMOBILIZED
FLUORESCEIN-GELATIN
41
ACKNOWLEDGMENTS This investigation was supported by Deutsche Forschungsgemeinschafi Grant De 18 l/9-5. The authors thank Prof. Dr. Greiling, Abteilung Klinische Chemie und Pathobiochemie, RWTH Aachen, for the opportunity to use his spectrofluorometer. or 0
20
LO
80
100
120
TIME6FIMIN~
2. Time course of digestion of FITC-labeled gelatin: 20 ~1 of FITC-labeled gelatin-Sephrose suspension and 10 ng trypsin in 580 1~10.05 M Tris-HCl buffer, pH 8.0, were incubated at 37°C with continuous shaking. After incubation time between 0 and 120 min, tubes were chilled on ice and centrifuged and the fluorescence of the supernatant was measured without background correction in a spectrofluorometer (excitation wavelength 480 nm, emission wavelength 5 18 nm).
REFERENCES
FIG.
same amount of FITC-labeled gelatin as 1 ng trypsin. The new assay is very simple, fast, and sufficiently reproducible. It has the advantage that no precipitation steps (2,4,7,15) or second enzyme reactions (4,10,11) are necessary. In comparison to a test with fluorescamine-labeled protein, the FITC-labeled substrate is remarkably stable and can be kept on stock rather then preparing the labeled substrate daily as necessary in the previous method ( 16). The stability of the substrate is comparable to a test with furanone-labeled substrates ( 17). Furthermore, if the described standard procedure is followed the tissue homogenate does not interfere with the detection of the fluorescence as in methods employing fluorescamine ( 18). Although no radioactive substrate is used, the test is as sensitive as a test with [3H]gelatin (7). It has proved useful for the detection of gelatinolytic activity in tissue samples. Furthermore, adaptation of the same test principle for the study of hydrolysis of other protein substrates labeled and immobilized in the same way should be possible.
1. Hemker, H. C. (ed.) (1983) in Handbook of Synthetic Substrates, pp. 27-47, Martinus Nijhoff, Boston. 2. McGillivray, D. H., Burnett, D., Afford, S. C., and Stockley, R. A. (1981) C/in. Chim Acta 111,289294.
Harris, E. D., and Cartwright, E. C. ( 1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J.. ed.), pp. 249-284, North-Holland, Amsterdam. 4. Wunderwald, P. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 258-270. Verlag Chemie, Weinheim. 5. Cheng, Y. S. E., and Aronson, A. I. (1977) Arch. Mi3.
crobiol.
115, 61-66.
6. Nelson, W. L., Ciaccio, E. I., and Hess, G. P. ( 196 1) Anal. Biochem.
2, 39-44.
7. Sunada, H., and Nagai, Y. (1980) J. Biochem. 87, 1765-1771. 8. Jensen, H. S., (1984) Biochem. J. 218,645-648. 9. Varani, J., Johnson, K., and Kaplan, J. (1980) Anal. Biochem.
107, 377-384.
10. Taylor, M. D., and Andrews, A. T. (1983) FEBSLett. 157,200-204.
11. Saunders, G. C., Svitra, Z., and Martinez, A. (1982) Anal. Biochem. 126, 122-l 30. 12. Sugiura, M., Ishikawa, M., Sasaki, M., Hirano, K., Ito, Y., and Awazu, S. (1979) Anal. Biochem. 97, 1l-16. 13. Cuatrecasas, P., Wilchek, M., and Anfinsen, C. B. (1968) Proc. Natl. Acad. Sci. USA 61, 636-643. 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (195 1) J. Biol. Chem. 193.265-275. 15. Twining, S. S. (1984) Anal. Biochem. 143, 30-34. 16. Ichihara, Y., Sogawa, K., and Takahashi, K. (1982) J. Biochem. 92, l-15. 17. Wiesner, R., and Troll, W. (1982) Anal. Biochem. 121, 290-294.
18. Evans, C. H., and Ridella, J. D. ( 1984) Anal. B&hem. 142,41
l-420.