Abstract
The temperature dependence of the heat capacity function of a recombinant streptokinase (rSK) has been studied by high-sensitivity differential scanning microcalorimetry and circular dichroism as a function of pH in low- and high-ionic strength buffers. At low ionic strength it is found that this protein, between pH 7 and 10, undergoes four reversible and independent two-state transitions during its unfolding, suggesting the existence of four domains in the native structure of the protein. This result reconciles previous conflicting reports about the number of domains of this protein obtained by differential scanning calorimetry and small-angle X-ray scattering. The number of two-state transitions decreases when the pH of the medium is decreased, without noticeable changes in its circular dichroism spectrum. A plausible localization of the four domains in the streptokinase sequences is proposed and their thermodynamic parameters are given. Increase of ionic strength to 200 mM NaCl affects positively the protein stability and confirms the existence of four reversible two-state transitions. Above 200 mM NaCl the protein stability decreases, resulting in low percentage of reversibility, and even irreversible transitions.
Similar content being viewed by others
REFERENCES
Baron, C., Freire, E., Mateo, P. L., and Cortijo, M. (1985). Acta Cient.Compost. 22, 303–317.
Biltonen, R. L. and Freire, E. (1978). Crit.Rev.Biochem. 5, 85–124.
Campos, M., Ortega, M., Padrón, G., Estrada, M. P., de la Fuente, J., and Herrera, L. (1992). Biotechniques 7, 638–641.
Castellino, F. J. (1983). Bioscience 33, 647–650.
Chibber, B. A. K., Morris, J. P., and Castellino, F. J. (1985). Biochemistry 24, 3429–3435.
Chibber, B. A. K., Radek, J. T., Morris, J. P., and Castellino, F. J. (1986). Proc.Natl.Acad.Sci.USA 83, 1237–1242.
Conejero-Lara, F., Parrado, J., Azuaga, A., Pointing, C. P., Smith, R. A. G., and Dobson, C. M. (1996). Protein Sci. 5, 2583–2591.
Conejero-Lara, F., Parrado, J., Azuaga, A., Dobson, C. M., and Pointing, C. P. (1998). Protein Sci. 7, 2190–2199.
Cortijo, M., Beldarrain, A., Molina, A. D., and López-Lacomba, J. L. (1995). Measurement Sci.Technol. 6, 1086–1092.
Damaschun, G., Damaschun, H., Gast, K., Gerlasch, D., Misselwitz, R., Welfle, H., and Zirwer, D. (1992). Eur.Biophys.J. 20, 355–361.
Estrada, M. P., Hérnández, L., Pérez, A., Rodriguez, P., Serrano, R., Rubiera, R., Pedraza, A., Padrón, G., Antuch, W., de la Fuente, J., and Herrera, L. (1992). Biotechnology 10, 1138–1142.
Freire, E. and Biltonen, R. L. (1978). Biopolymers 17, 463–479.
Gill, S. C. and von Hippel, P. H. (1989). Anal.Chem. 182, 319–326.
Hernandez, L., Rodriguez, P., Castro, A., Serrano, R., Rodriguez, M. P., Rubiera, R., Estada, M. P., Pérez, A., de la Fuente, J., and Herrera L. (1990). Biotechnologia Aplicada 7, 53–160.
Huang T., Malke, H., and Ferreti, J. (1989). Mol.Biol. 2, 197–205.
López-Lacomba, J. L., Cortijo, M., Guzman, M., Mateo, P. L., Aguirre, R., Harvey, S., and Cheung, H. C. (1989). Biopolymers 28, 2143–2159.
Makhatadze, G. I. and Privalov, P. L. (1990). J.Mol.Biol. 213, 375–384.
Makhatadze, G. I., Medvedkin, V. N., and Privalov, P. L. (1990). Biopolymers 30, 1001–1010.
Medved, L. V., Solovjov, D. A., and Inghain, C. I. (1996). Eur.J.Biochem. 239, 333–339.
Misselwitz, R., Kraft, R., Kostka, H., Fabian, K., Welfle, K., Pfeil, W., Welfle, H., and Gerlach, D. (1992). Int.J.Biol.Macromol. 14, 107–116.
Parrado, J., Conejero-Lara, F., Smith, R. A. G., Marshall, J., Pointing, C. P., and Dobson, C. M. (1996). Protein Sci. 5, 693–704.
Perczel, A., Park, K., and Fasman, G.-D. (1992). Anal.Biochem. 203, 83–93. Privalov, P. L. and Makhatadze, G. I. (1990). J.Mol.Biol. 213, 385–391. Radek, J. T. and Castellino, F. J. (1989). J.Biol.Chem. 264, 9915–9922. Radek, J. T., Davidson, D. J., and Castellino, F. J. (1993). Meth.Enzymol. 223, 45–155.
Rodriguez, P., Fuentes, P., Muñoz, E., Orta, D., Alburquerque, S., Perez, S., Vesada, V., and Herrera, L. (1994). Fibrinilysis 8, 276–285.
Rodriguez, P., Fuentes, P., Barro, M., Alvarez, J. G., Muñoz, E., Collen, D., and Lijnen, H. R. (1985). Eur.J.Biochem. 229, 83–90.
Sanchez-Ruiz, J. M. (1992). Biophys.J. 61, 921–935.
Siefring, G. E. and Castellino, F. J. (1976). J.Biol.Chem. 251, 3913–3920.
Takahashi, K., and Sturtevant, J. M. (1981). Biochemistry 20, 6185–6190.
Teuten, A. J., Broadhurst, R. W., Smith, R. A. G., and Dobson, C. M. (1993). Biochem, J. 290, 313–319.
Wang, X., Lin, X., Loy, A., Tang, J., and Zhang, X. (1998). Science 281, 1662–1665.
Welfle, H., Misselwitz, R., Fabian, H., Damerau, W., Hoelzer, W., Gerlach, D., Kalnin, N. N., and Venyaminov, S. Y. (1992a). Int.J.Biol.Macromol. 14, 9–18.
Welfle, K., Pfeil, W., Misselwitz, R., Welfle, H., and Gerlach, D. (1992b). Int.J.Biol.Macromol. 14, 19–22.
Welfle, K., Misselwitz, R., Schaup, A., Gerlach, D., and Welfle, H. (1997). Protein Struc.Funct.Genet. 27, 26–35.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Beldarraín, A., López-Lacomba, J.L., Kutyshenko, V.P. et al. Multidomain Structure of a Recombinant Streptokinase. A Differential Scanning Calorimetry Study. J Protein Chem 20, 9–17 (2001). https://doi.org/10.1023/A:1011044718840
Published:
Issue Date:
DOI: https://doi.org/10.1023/A:1011044718840