Abstract
Cloning of the thrombin cDNA has made it possible to study thrombin function by site-directed mutagenesis. Quantitative results from studies of thrombin mutants are often hindered by difficulties in assaying the enzyme activity. The high enzyme concentrations required for activity determination by standard methods limit their usefulness to thrombin mutants that cannot be readily produced in large quantities. We have developed a novel method using the synthetic substrate S-2238 and hirudin, a tight-binding inhibitor of thrombin, that allows for the active-site titration of thrombin at concentrations as low as 20 pM, with an error of ≤5%. In addition, hirudin activity can be determined by this method to concentrations as low as 40 pM, with an error of ≤5%.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ayala, Y., and Di Cera, E. (1994).J. Mol. Biol. 235, 733–746.
Cha, S. (1976).Biochem. Pharmacol. 25, 2695–2702.
Chase, T., Jr., and Shaw, E. (1969).Biochemistry 8, 2212–2224.
Davie, E. W., Fujikawa, K., and Kisiel, W. (1991).Biochemistry 30, 10363–10370.
Degen, S. J. F., MacGillivray, R. T. A., and Davie, E. W. (1983).Biochemistry 22, 2087–2097.
Degryse, E. (1990).Thromb. Res. 60, 433–443.
Fenton, J. W., II, Fasco, M. G., Stackrow, A. B., Aronson, D. L., Young, A. M., and Finlayson, J. S. (1977).J. Biol. Chem. 252, 3587–3598.
Hitomi, Y., Kanda, T., Niinobe, M., and Fujii, S. (1981).Clin. Chim. Acta 119, 157–164.
Hopfner, K.-P., Ayala, Y., Szewczuk, Z., Konishi, Y., and Di Cera, E. (1993).Biochemistry 32, 2947–2953.
Jameson, G. W., Roberts, D. V., Adams, R. W., Kyle, W. S. A., and Elmore, D. T. (1973).Biochem. J. 131, 107–117.
Jorpes, E., Vrethammar, T., ÖHman, B., and Blombäck, B. (1958).J. Pharmacy Pharmacol. 10, 561–573.
Le Bonniec, B., and Esmon, C. T. (1991).Proc. Natl. Acad. Sci. USA 88, 7371–7375.
Markwardt, F. (1970).Meth. Enzymol. 19, 924–932.
Stone, S., and Maraganore, J. M. (1993).Meth. Enzymol. 223, 312–336.
Veharand, G. A., and Davie, E. W. (1980).Biochemistry 19, 401–409.
Wells, C., and Di Cera, E. (1992).Biochemistry 31, 11721–11730.
Wu, Q., Sheehan, J. P., Tsiang, M., Lentz, S. R., Birktoft, J. J., and Sadler, J. E. (1991).Proc. Natl. Acad. Sci. USA 88, 6775–6779.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Dang, Q.D., Di Cera, E. A simple activity assay for thrombin and hirudin. J Protein Chem 13, 367–373 (1994). https://doi.org/10.1007/BF01901692
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01901692