- Email: [email protected]
Electrophoretic Analysis of Protease Inhibitors in Fibrin Zymography
179
NOTES & TIPS
In conclusion, 2-D gels preserved for almost two decades can still serve as a source material for protein identification by in-gel digestion and MALDI-TOF. MALDI-TOF with delayed extraction enabled us to observe peptide fragments at isotopic resolution, rendering the assignment of the protein through a database search highly reliable. The use of once dried and archived gels for protein analysis by mass spectrometry will provide an opportunity to reexamine experimental results achieved many years ago for confirmation and/or further investigation. The method shown here will become more useful as genome databases for human and some model animals are completed in the near future (19). Acknowledgments. This work was supported by NIH Grant EY06595 and Oklahoma Center for the Advancement of Science and Technology (OCAST) Awards HR3-080 to H.M. and HN5-024 to N.K. The 2-D gels shown in this work had been run in the project supported by an NSF grant (BNS 83 15599) to William L. Pak and H.M. The PerSeptive Biosytems Voyager Elite laser desorption ionization time-of-flight mass spectrometer was purchased by the NSF EPSCoR Oklahoma Biotechnology Network grant. We thank Masaomi Matsumoto for reading the manuscript.
REFERENCES 1. 2. 3. 4.
5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
O’Farrell, P. H. (1975) J. Biol. Chem. 250, 4007– 4021. Fenselau, C. (1997) Anal. Chem. 69, 661A– 665A. Yates, J. R. 3rd (1998) J. Mass Spectrom. 33, 1–19. Matsumoto, H., Kurien, B. T., Takagi, Y., Kahn, E. S., Kinumi, T., Komori, N., Yamada, T., Hayashi, F., Isono, K., Pak, W. L., Jackson, K. W., and Tobin, S. L. (1994) Neuron 12, 997– 1010. Kinumi, T., Tobin, S. L., Matsumoto, H., Jackson, K. W., and Ohashi, M. (1997) Eur. Mass Spectrom. 3, 367–378. Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Boucherie, H., and Mann, M. (1996) Proc. Natl. Acad. Sci. USA 93, 14440 –14445. Li, G., Waltham, M., Anderson, N. L., Unsworth, E., Treston, A., and Weinstein, J. N. (1997) Electrophoresis 18, 391– 402. Larsson, T., Norbeck, J., Karlsson, H., and Blomberg, A. (1997) Electrophoresis 18, 418 – 423. Qiu, Y., Benet, L. Z., and Burlingame, A. L. (1998) J. Biol. Chem. 271, 17940 –17953. Nishizawa, Y., Jackson, K. W., Usukura, J., Tobin, S. L. and Matsumoto, H. (1996) Invest. Ophthalmol. Visual Sci. 73, S637. Matsumoto, H., O’Tousa, J., and Pak, W. L. (1982) Science 217, 839 – 841. Matsumoto, H., and Pak, W. L. (1984) Science 223, 184 –186. Rosenfeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992) Anal. Biochem. 203, 173–179. Pappin, D. J. C., Hojrup, P., and Bleasby, A. J. (1993) Curr. Biol. 3, 327–332. Reinke, R., Krantz, D. E., Yen, D., and Zipursky, S. L. (1988) Cell 52, 291–301. Fujita, S. C., Zipursky, S. L., Benzer, S., Ferrus, A., and Shotwell, S. L. (1982). Proc. Natl. Acad. Sci. USA 79, 7929 –7933. Fujita, S. C., Inoue, H., Yoshioka, T., and Hotta, Y. (1987) Biochem. J. 243, 97–104.
18. Shevchenko, A., Shevchenko, A., Schraven, B., and Mann, M. (1998) The 46th ASMS Conference on Mass Spectrometry and Allied Topics, p. 238. [Abstract] 19. Waterston, R., and Sulston, J. E. (1998) Science 282, 53–54.
Electrophoretic Analysis of Protease Inhibitors in Fibrin Zymography Seung-Ho Kim 1 and Nack-Shick Choi Protein Function Research Unit, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea Received December 29, 1998
Zymography techniques are used routinely to identify proteolytic activity in polyacrylamide gels under nonreducing conditions (1). These methods are based on a SDS–polyacrylamide gel copolymerized with a protein substrate such as gelatin (1–3), casein (4), or fibrin (5) that is degraded by the proteases restored during the incubation period in enzyme reaction buffer after the electrophoretic separation. We established a new fibrin zymography method that can identify the enzymes using enzyme reaction buffer containing protease inhibitors such as PMSF 2 (a known inhibitor of serine proteases) or EDTA (a known inhibitor of metalloproteases). This new zymography technique gave several benefits. First, we could classify and also identify the active serine proteases and metalloproteases among the protein pools. This makes it possible to determine which protein contains activity and is going to be isolated before starting protein purification. Furthermore, it would be possible to verify unknown protease inhibitors to specific proteolytic enzymes. Separating gel solution (12%, w/v) containing fibrinogen (0.12%, w/v, 0.15 sodium salt) was prepared in a manner similar to that described by Kim et al. (5) in a total volume of 10 mL and centrifuged to remove insoluble impurities which were induced when SDS stock solution was mixed. Thrombin (1 NIH unit/mL) solution was added to the gel solution in final concentration of 0.1 munit/mL. Ammonium persulfate (0.04% w/v) and N,N,N9,N9-tetramethylethylenediamine (0.028%, v/v) were used to catalyze the polymerization. One hundred nanograms of plasmin in zymogram sample buffer (53) consisting of 0.5 M Tris, pH 6.8, 20% SDS, 20% glycerol, and 0.03% bromphenol blue was electro1 To whom correspondence should be addressed. Fax: 82-42-8604593. E-mail: [email protected]. 2 Abbreviation used: PMSF, phenylmethylsulfonyl fluoride.
Analytical Biochemistry 270, 179 –181 (1999) Article ID abio.1999.4080 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
180
NOTES & TIPS
FIG. 1. Inhibition of plasmin in the fibrin zymography. 100 ng of plasmin was electrophoresed into the fibrin-copolymerized gel at 15 mA constant currency and subsequently soaked in 50 mM of Tris buffer (pH 7.4) containing 2.5% Triton X-100 for 30 min to remove SDS. After removal of Triton X-100 with distillation water, the gel was incubated in a reaction buffer (30 mM Tris, pH 7.4, 200 mM NaCl, and 0.02% NaN 3) at 37°C for 15 h. In the inhibition test, indicated concentrations of PMSF (A) and EDTA (B) were added to the enzyme reaction buffer.
phoresed according to the method of Laemmli (6) into the fibrin gel and subsequently soaked in 2.5% Triton X-100 solution for 30 min and then incubated in a reaction buffer (30 mM Tris, pH 7.4, 200 mM NaCl, and 0.02% NaN 3) at 37°C for 15 h as previously described (1). In the case of the inhibition test using zymography, various concentrations of PMSF or EDTA were added in the enzyme reaction buffer. Figure 1 shows the fibrin zymography in which plasmin appears as clear bands of fibrinolysis against a blue background of undigested fibrin substrate. The plasmin activity was inhibited about 90% by 5 mM PMSF and almost inhibited by 10 mM of PMSF but not was affected by EDTA (Fig. 1). We have also measured the amidolytic activity of the plasmin with Beckman DU-70 spectrophotometer using a chromogenic substrate (N-p-tosyl-Gly-Pro-LyspNA, T-6140) (7). To test whether the PMSF and EDTA can inhibit the plasmin activity, the mixture of plasmin (3 mg/0.1 mL) and 0.1 mL of various concentration of inhibitors as indicated in the Table 1 was incubated at 37°C for 10 min. Assays were performed in 20 mM sodium phosphate buffer (pH 7.0), 0.2 mL of 0.5 mM substrate and plasmin (3 mg/0.2 mL). The mixture was incubated at 37°C in a water bath for 1 min and the reaction was stopped by adding 0.1 mL of 50% acetic acid. The activity was determined from the absorbance at 405 nm as the formation of p-nitroaniline. One unit was defined as the amount of substrate hydrolyzed per
TABLE 1
The Effect of Inhibitors of the Plasmin Activity Using the Substrate (T-6140) a Inhibitors (mM)
0
1
5
10
PMSF EDTA
100 100
37.3 98.9
21.8 98.2
8.8 96.1
a
T-6140: substrate (N-p-tosyl-Gly-Lys-pNA).
FIG. 2. Electrophoretic analysis of protease inhibitor in the fibrin zymography. The assay was carried out with the culture medium of Bacillus sp. strain (designated as DJ-4) isolated from Doen-Jang, a traditional Korean fermented food. 100 ng of the crude enzyme solution, which was obtained by centrifugation at 10,000g for 10 min, was electrophoresed as described in the legend to Fig. 1. The inhibition assay was performed without inhibitor (1), with 5 mM PMSF (2), and with EDTA (3).
minute per milliliter by the enzyme. The plasmin activity was inhibited about 80% by 5 mM PMSF, but was not influenced significantly by EDTA (Table 1). We have examined whether the fibrin zymography is acceptable for enzyme activity inhibitor assays of the culture medium. To identify this possibility, we prepared culture media samples from the Bacillus sp. strain isolated from Doen-Jang, a traditional Korean fermented food (Fig. 2). It was well known that Bacillus sp. strains produce various extracellular proteases such as an alkaline protease, a neutral metalloprotease, and an esterase (7). We could identify the four major proteases secreted by Bacillus sp. in fibrin zymography (Fig. 2). Two bands (53 and 64 kDa) were inhibited by 5 mM EDTA (Fig. 2, lane 3), the 29-kDa band was affected by PMSF (Fig. 2, lane 2), and both PMSF and EDTA inhibited the 38-kDa band (Fig. 2, lanes 2 and 3). Given this information we could tentatively classify the enzymes with apparent molecular mass of 53 and 64 kDa as metalloproteases (Fig. 2) and the 29-kDa band as a serine protease (Fig. 2) without enzyme purification. We believe that this new method will be helpful for purification and characterization of the enzymes. REFERENCES 1. Kleiner, D. E., and Stetler-Stevenson, W. G. (1994) Anal. Biochem. 218, 325–329. 2. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196 –202. 3. Leber, T. M., and Balkwill, F. R. (1997) Anal. Biochem. 249, 24 –28. 4. Raser, K. J., Posner, A., and Wang, K. K. W. (1995) Arch. Biochem. Biophys. 319, 211–216. 5. Kim, S. H., Choi, N. S., and Lee, W. Y. (1998) Anal. Biochem. 263, 115–116. 6. Laemmli, U. K. (1970) Nature 227, 680 – 685.
NOTES & TIPS 7. Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I., and Lee, S.(1996) Appl. Environ. Microbiol. 62, 2482– 2488.
Determination of Phosphate and Pyrophosphate Ions by Capillary Electrophoresis Odile He´nin, Bernard Barbier, and Andre´ Brack 1 Centre de Biophysique Mole´culaire, CNRS, rue Charles Sadron, 45071 Orle´ans cedex 2, France Received January 12, 1999
Inorganic pyrophosphatase is an enzyme known to catalyze the hydrolysis of pyrophosphate ions (PPi) 2 into two phosphate ions (Pi) and to require divalent metallic cations for its full activity (1– 4). Pyrophosphate hydrolysis is usually assayed by measuring the release of Pi by the colorimetric method originally introduced by Fiske and Subbarow (5). Although this method is very sensitive, it does not allow the direct determination of PPi. The same limitation holds for the multistep coupled enzyme assays (6 –10). Recently, capillary electrophoresis (CE) coupled with indirect uv detection was introduced for the separation of poorly uv-absorbing polyanions. Chromophores with appropriate mobility are used in association with quaternary ammonium ions to obtain a dynamic coating of the fused-silica capillary neutralizing the electroosmotic flow (11–13). We describe a fast and simple method for the simultaneous assay of PPi and Pi based on their separation by capillary electrophoresis in buffer containing metallic ions mimicking biological milieu. We used indirect uv detection of the ATP electrophoretic chromophore at 254 nm and CTAB as a electroosmotic flow modifier as previously recommended by Wang and Li (14) for the separation of polyphosphates. ATP has a strong uv absorption at 254 nm and its mobility was found to be between that of PPi and Pi. The method has been shown to be extensible to various media including pure water and salt-containing aqueous solutions (MgCl 2, ZnCl 2, or NaF). 1 To whom correspondence should be addressed. Fax: 33-2-38-6315-17. E-mail: [email protected]. 2 Abbreviation used: PPi, pyrophosphate; PPase, inorganic pyrophosphatase; Pi, phosphate; Mops, 3-morpholinopropanesulfonic acid; ATP, adenosine triphosphate; CTAB, hexadecyltrimethyl-ammonium bromide; CE, capillary electrophoresis.
Analytical Biochemistry 270, 181–184 (1999) Article ID abio.1999.4068 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
181
MATERIALS AND METHODS
Reagents Lyophilized yeast inorganic pyrophosphatase (EC 3.6.1.1) containing 85% buffer salts, ATP disodium salt, ammonium molybdate tetrahydrate, and malachite green oxalate salt was purchased from Sigma. Hydrochloric acid (trace select grade) was purchased from Fluka Chemika. Na 4P 2O 7,10H 2O; Na 2HPO 4, 12H 2O, MgCl 2,6H 2O (microselect grade), ZnCl 2, Mops, CTAB, and EDTA were purchased from Fluka Biochemika. Water for chromatography was purchased from Merck. Capillary Electrophoresis Procedures Simultaneous assays of PPi and Pi were performed with a Beckman P/ACE System 5000 capillary electrophoresis setup equipped with an uv/vis detector. Two types of capillaries were used. Polyimide-coated fusedsilica capillaries of i.d. 75 mm and o.d. 375 mm from Beckman were used when dynamic coating with CTAB was used. The total length was 47 cm with an effective length of 40 cm. The capillary was successively flushed with 1 M HCl for 10 min, 0.1 M NaOH for 10 min, water for 5 min, and finally with buffer for 30 min every day prior to CE measurements. Between two successive runs, the capillary was flushed with buffer for 2 min. Before storage, the capillary was rinsed with water for 10 min. Commercially precoated fused-silica capillaries (CElect-N from Supelco of i.d. 75 mm and o.d. 360 mm) were used when a static coating was considered. The total length was 22 cm with an effective length of 15 cm. The capillary was conditioned daily with water and then with buffer for 30 min. The capillary was flushed with buffer for 2 min between two successive runs and rinsed with water before storage. Buffer. Buffer (10 mM ATP and 0.05 mM CTAB) was prepared and adjusted to pH 7.2 with 0.1 M NaOH for the fused-silica capillary coated with CTAB. With the precoated capillary CElect-N, a 10 mM ATP buffer was prepared and adjusted to pH 7.2 with 0.1 M NaOH. CE assay of PPi and Pi. Sample solutions containing various amounts of both PPi and Pi were injected into the capillary at the cathodic side by hydrodynamic injection mode with an injection time of 5 s unless otherwise stated. The separation voltage was 30 kV with the fused-silica capillary coated with CTAB and 15 kV for the precoated CElect-N capillary. The temperature was kept at 25°C. Postcolumn indirect uv detection was conducted at 254 nm. A complete analytical run required 3.5 min. Calibration curves. Different solutions containing both Pi up to 1.6 mM and PPi up to 0.8 mM in 40 mM Mops buffer, pH 7.2, and 0.8 mM MgCl 2, mimicking the
NOTES & TIPS
In conclusion, 2-D gels preserved for almost two decades can still serve as a source material for protein identification by in-gel digestion and MALDI-TOF. MALDI-TOF with delayed extraction enabled us to observe peptide fragments at isotopic resolution, rendering the assignment of the protein through a database search highly reliable. The use of once dried and archived gels for protein analysis by mass spectrometry will provide an opportunity to reexamine experimental results achieved many years ago for confirmation and/or further investigation. The method shown here will become more useful as genome databases for human and some model animals are completed in the near future (19). Acknowledgments. This work was supported by NIH Grant EY06595 and Oklahoma Center for the Advancement of Science and Technology (OCAST) Awards HR3-080 to H.M. and HN5-024 to N.K. The 2-D gels shown in this work had been run in the project supported by an NSF grant (BNS 83 15599) to William L. Pak and H.M. The PerSeptive Biosytems Voyager Elite laser desorption ionization time-of-flight mass spectrometer was purchased by the NSF EPSCoR Oklahoma Biotechnology Network grant. We thank Masaomi Matsumoto for reading the manuscript.
REFERENCES 1. 2. 3. 4.
5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
O’Farrell, P. H. (1975) J. Biol. Chem. 250, 4007– 4021. Fenselau, C. (1997) Anal. Chem. 69, 661A– 665A. Yates, J. R. 3rd (1998) J. Mass Spectrom. 33, 1–19. Matsumoto, H., Kurien, B. T., Takagi, Y., Kahn, E. S., Kinumi, T., Komori, N., Yamada, T., Hayashi, F., Isono, K., Pak, W. L., Jackson, K. W., and Tobin, S. L. (1994) Neuron 12, 997– 1010. Kinumi, T., Tobin, S. L., Matsumoto, H., Jackson, K. W., and Ohashi, M. (1997) Eur. Mass Spectrom. 3, 367–378. Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Boucherie, H., and Mann, M. (1996) Proc. Natl. Acad. Sci. USA 93, 14440 –14445. Li, G., Waltham, M., Anderson, N. L., Unsworth, E., Treston, A., and Weinstein, J. N. (1997) Electrophoresis 18, 391– 402. Larsson, T., Norbeck, J., Karlsson, H., and Blomberg, A. (1997) Electrophoresis 18, 418 – 423. Qiu, Y., Benet, L. Z., and Burlingame, A. L. (1998) J. Biol. Chem. 271, 17940 –17953. Nishizawa, Y., Jackson, K. W., Usukura, J., Tobin, S. L. and Matsumoto, H. (1996) Invest. Ophthalmol. Visual Sci. 73, S637. Matsumoto, H., O’Tousa, J., and Pak, W. L. (1982) Science 217, 839 – 841. Matsumoto, H., and Pak, W. L. (1984) Science 223, 184 –186. Rosenfeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992) Anal. Biochem. 203, 173–179. Pappin, D. J. C., Hojrup, P., and Bleasby, A. J. (1993) Curr. Biol. 3, 327–332. Reinke, R., Krantz, D. E., Yen, D., and Zipursky, S. L. (1988) Cell 52, 291–301. Fujita, S. C., Zipursky, S. L., Benzer, S., Ferrus, A., and Shotwell, S. L. (1982). Proc. Natl. Acad. Sci. USA 79, 7929 –7933. Fujita, S. C., Inoue, H., Yoshioka, T., and Hotta, Y. (1987) Biochem. J. 243, 97–104.
18. Shevchenko, A., Shevchenko, A., Schraven, B., and Mann, M. (1998) The 46th ASMS Conference on Mass Spectrometry and Allied Topics, p. 238. [Abstract] 19. Waterston, R., and Sulston, J. E. (1998) Science 282, 53–54.
Electrophoretic Analysis of Protease Inhibitors in Fibrin Zymography Seung-Ho Kim 1 and Nack-Shick Choi Protein Function Research Unit, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea Received December 29, 1998
Zymography techniques are used routinely to identify proteolytic activity in polyacrylamide gels under nonreducing conditions (1). These methods are based on a SDS–polyacrylamide gel copolymerized with a protein substrate such as gelatin (1–3), casein (4), or fibrin (5) that is degraded by the proteases restored during the incubation period in enzyme reaction buffer after the electrophoretic separation. We established a new fibrin zymography method that can identify the enzymes using enzyme reaction buffer containing protease inhibitors such as PMSF 2 (a known inhibitor of serine proteases) or EDTA (a known inhibitor of metalloproteases). This new zymography technique gave several benefits. First, we could classify and also identify the active serine proteases and metalloproteases among the protein pools. This makes it possible to determine which protein contains activity and is going to be isolated before starting protein purification. Furthermore, it would be possible to verify unknown protease inhibitors to specific proteolytic enzymes. Separating gel solution (12%, w/v) containing fibrinogen (0.12%, w/v, 0.15 sodium salt) was prepared in a manner similar to that described by Kim et al. (5) in a total volume of 10 mL and centrifuged to remove insoluble impurities which were induced when SDS stock solution was mixed. Thrombin (1 NIH unit/mL) solution was added to the gel solution in final concentration of 0.1 munit/mL. Ammonium persulfate (0.04% w/v) and N,N,N9,N9-tetramethylethylenediamine (0.028%, v/v) were used to catalyze the polymerization. One hundred nanograms of plasmin in zymogram sample buffer (53) consisting of 0.5 M Tris, pH 6.8, 20% SDS, 20% glycerol, and 0.03% bromphenol blue was electro1 To whom correspondence should be addressed. Fax: 82-42-8604593. E-mail: [email protected]. 2 Abbreviation used: PMSF, phenylmethylsulfonyl fluoride.
Analytical Biochemistry 270, 179 –181 (1999) Article ID abio.1999.4080 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
180
NOTES & TIPS
FIG. 1. Inhibition of plasmin in the fibrin zymography. 100 ng of plasmin was electrophoresed into the fibrin-copolymerized gel at 15 mA constant currency and subsequently soaked in 50 mM of Tris buffer (pH 7.4) containing 2.5% Triton X-100 for 30 min to remove SDS. After removal of Triton X-100 with distillation water, the gel was incubated in a reaction buffer (30 mM Tris, pH 7.4, 200 mM NaCl, and 0.02% NaN 3) at 37°C for 15 h. In the inhibition test, indicated concentrations of PMSF (A) and EDTA (B) were added to the enzyme reaction buffer.
phoresed according to the method of Laemmli (6) into the fibrin gel and subsequently soaked in 2.5% Triton X-100 solution for 30 min and then incubated in a reaction buffer (30 mM Tris, pH 7.4, 200 mM NaCl, and 0.02% NaN 3) at 37°C for 15 h as previously described (1). In the case of the inhibition test using zymography, various concentrations of PMSF or EDTA were added in the enzyme reaction buffer. Figure 1 shows the fibrin zymography in which plasmin appears as clear bands of fibrinolysis against a blue background of undigested fibrin substrate. The plasmin activity was inhibited about 90% by 5 mM PMSF and almost inhibited by 10 mM of PMSF but not was affected by EDTA (Fig. 1). We have also measured the amidolytic activity of the plasmin with Beckman DU-70 spectrophotometer using a chromogenic substrate (N-p-tosyl-Gly-Pro-LyspNA, T-6140) (7). To test whether the PMSF and EDTA can inhibit the plasmin activity, the mixture of plasmin (3 mg/0.1 mL) and 0.1 mL of various concentration of inhibitors as indicated in the Table 1 was incubated at 37°C for 10 min. Assays were performed in 20 mM sodium phosphate buffer (pH 7.0), 0.2 mL of 0.5 mM substrate and plasmin (3 mg/0.2 mL). The mixture was incubated at 37°C in a water bath for 1 min and the reaction was stopped by adding 0.1 mL of 50% acetic acid. The activity was determined from the absorbance at 405 nm as the formation of p-nitroaniline. One unit was defined as the amount of substrate hydrolyzed per
TABLE 1
The Effect of Inhibitors of the Plasmin Activity Using the Substrate (T-6140) a Inhibitors (mM)
0
1
5
10
PMSF EDTA
100 100
37.3 98.9
21.8 98.2
8.8 96.1
a
T-6140: substrate (N-p-tosyl-Gly-Lys-pNA).
FIG. 2. Electrophoretic analysis of protease inhibitor in the fibrin zymography. The assay was carried out with the culture medium of Bacillus sp. strain (designated as DJ-4) isolated from Doen-Jang, a traditional Korean fermented food. 100 ng of the crude enzyme solution, which was obtained by centrifugation at 10,000g for 10 min, was electrophoresed as described in the legend to Fig. 1. The inhibition assay was performed without inhibitor (1), with 5 mM PMSF (2), and with EDTA (3).
minute per milliliter by the enzyme. The plasmin activity was inhibited about 80% by 5 mM PMSF, but was not influenced significantly by EDTA (Table 1). We have examined whether the fibrin zymography is acceptable for enzyme activity inhibitor assays of the culture medium. To identify this possibility, we prepared culture media samples from the Bacillus sp. strain isolated from Doen-Jang, a traditional Korean fermented food (Fig. 2). It was well known that Bacillus sp. strains produce various extracellular proteases such as an alkaline protease, a neutral metalloprotease, and an esterase (7). We could identify the four major proteases secreted by Bacillus sp. in fibrin zymography (Fig. 2). Two bands (53 and 64 kDa) were inhibited by 5 mM EDTA (Fig. 2, lane 3), the 29-kDa band was affected by PMSF (Fig. 2, lane 2), and both PMSF and EDTA inhibited the 38-kDa band (Fig. 2, lanes 2 and 3). Given this information we could tentatively classify the enzymes with apparent molecular mass of 53 and 64 kDa as metalloproteases (Fig. 2) and the 29-kDa band as a serine protease (Fig. 2) without enzyme purification. We believe that this new method will be helpful for purification and characterization of the enzymes. REFERENCES 1. Kleiner, D. E., and Stetler-Stevenson, W. G. (1994) Anal. Biochem. 218, 325–329. 2. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196 –202. 3. Leber, T. M., and Balkwill, F. R. (1997) Anal. Biochem. 249, 24 –28. 4. Raser, K. J., Posner, A., and Wang, K. K. W. (1995) Arch. Biochem. Biophys. 319, 211–216. 5. Kim, S. H., Choi, N. S., and Lee, W. Y. (1998) Anal. Biochem. 263, 115–116. 6. Laemmli, U. K. (1970) Nature 227, 680 – 685.
NOTES & TIPS 7. Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I., and Lee, S.(1996) Appl. Environ. Microbiol. 62, 2482– 2488.
Determination of Phosphate and Pyrophosphate Ions by Capillary Electrophoresis Odile He´nin, Bernard Barbier, and Andre´ Brack 1 Centre de Biophysique Mole´culaire, CNRS, rue Charles Sadron, 45071 Orle´ans cedex 2, France Received January 12, 1999
Inorganic pyrophosphatase is an enzyme known to catalyze the hydrolysis of pyrophosphate ions (PPi) 2 into two phosphate ions (Pi) and to require divalent metallic cations for its full activity (1– 4). Pyrophosphate hydrolysis is usually assayed by measuring the release of Pi by the colorimetric method originally introduced by Fiske and Subbarow (5). Although this method is very sensitive, it does not allow the direct determination of PPi. The same limitation holds for the multistep coupled enzyme assays (6 –10). Recently, capillary electrophoresis (CE) coupled with indirect uv detection was introduced for the separation of poorly uv-absorbing polyanions. Chromophores with appropriate mobility are used in association with quaternary ammonium ions to obtain a dynamic coating of the fused-silica capillary neutralizing the electroosmotic flow (11–13). We describe a fast and simple method for the simultaneous assay of PPi and Pi based on their separation by capillary electrophoresis in buffer containing metallic ions mimicking biological milieu. We used indirect uv detection of the ATP electrophoretic chromophore at 254 nm and CTAB as a electroosmotic flow modifier as previously recommended by Wang and Li (14) for the separation of polyphosphates. ATP has a strong uv absorption at 254 nm and its mobility was found to be between that of PPi and Pi. The method has been shown to be extensible to various media including pure water and salt-containing aqueous solutions (MgCl 2, ZnCl 2, or NaF). 1 To whom correspondence should be addressed. Fax: 33-2-38-6315-17. E-mail: [email protected]. 2 Abbreviation used: PPi, pyrophosphate; PPase, inorganic pyrophosphatase; Pi, phosphate; Mops, 3-morpholinopropanesulfonic acid; ATP, adenosine triphosphate; CTAB, hexadecyltrimethyl-ammonium bromide; CE, capillary electrophoresis.
Analytical Biochemistry 270, 181–184 (1999) Article ID abio.1999.4068 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
181
MATERIALS AND METHODS
Reagents Lyophilized yeast inorganic pyrophosphatase (EC 3.6.1.1) containing 85% buffer salts, ATP disodium salt, ammonium molybdate tetrahydrate, and malachite green oxalate salt was purchased from Sigma. Hydrochloric acid (trace select grade) was purchased from Fluka Chemika. Na 4P 2O 7,10H 2O; Na 2HPO 4, 12H 2O, MgCl 2,6H 2O (microselect grade), ZnCl 2, Mops, CTAB, and EDTA were purchased from Fluka Biochemika. Water for chromatography was purchased from Merck. Capillary Electrophoresis Procedures Simultaneous assays of PPi and Pi were performed with a Beckman P/ACE System 5000 capillary electrophoresis setup equipped with an uv/vis detector. Two types of capillaries were used. Polyimide-coated fusedsilica capillaries of i.d. 75 mm and o.d. 375 mm from Beckman were used when dynamic coating with CTAB was used. The total length was 47 cm with an effective length of 40 cm. The capillary was successively flushed with 1 M HCl for 10 min, 0.1 M NaOH for 10 min, water for 5 min, and finally with buffer for 30 min every day prior to CE measurements. Between two successive runs, the capillary was flushed with buffer for 2 min. Before storage, the capillary was rinsed with water for 10 min. Commercially precoated fused-silica capillaries (CElect-N from Supelco of i.d. 75 mm and o.d. 360 mm) were used when a static coating was considered. The total length was 22 cm with an effective length of 15 cm. The capillary was conditioned daily with water and then with buffer for 30 min. The capillary was flushed with buffer for 2 min between two successive runs and rinsed with water before storage. Buffer. Buffer (10 mM ATP and 0.05 mM CTAB) was prepared and adjusted to pH 7.2 with 0.1 M NaOH for the fused-silica capillary coated with CTAB. With the precoated capillary CElect-N, a 10 mM ATP buffer was prepared and adjusted to pH 7.2 with 0.1 M NaOH. CE assay of PPi and Pi. Sample solutions containing various amounts of both PPi and Pi were injected into the capillary at the cathodic side by hydrodynamic injection mode with an injection time of 5 s unless otherwise stated. The separation voltage was 30 kV with the fused-silica capillary coated with CTAB and 15 kV for the precoated CElect-N capillary. The temperature was kept at 25°C. Postcolumn indirect uv detection was conducted at 254 nm. A complete analytical run required 3.5 min. Calibration curves. Different solutions containing both Pi up to 1.6 mM and PPi up to 0.8 mM in 40 mM Mops buffer, pH 7.2, and 0.8 mM MgCl 2, mimicking the