Abstract
Moderate temperatures or low concentrations of denaturants diminish the catalytic activity of some enzymes before spectroscopic methods indicate protein unfolding. To discriminate between possible reasons for the inactivation of ribonuclease A, we investigated the influence of temperature and guanidine hydrochloride on its proteolytic susceptibility to proteinase K by determining the proteolytic rate constants and fragment patterns. The results were related to changes of activity and spectroscopic properties of ribonuclease A. With thermal denaturation, the changes in activity and in the rate constants of proteolytic degradation coincide and occur slightly before the spectroscopically observable transition. In the case of guanidine hydrochloride-induced denaturation, however, proteolytic resistance of ribonuclease A initially increases accompanied by a drastic activity decrease far before unfolding of the protein is detected by spectroscopy or proteolysis. In addition to ionic effects, a tightening of the protein structure at low guanidine hydrochloride concentrations is suggested to be responsible for ribonuclease A inactivation.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
REFERENCES
Anfinsen, C. B. (1972). Biochem. J. 128, 737–749.
Arnold, U. and Ulbrich-Hofmann, R. (1997). Biochemistry 36, 2166–2172.
Arnold, U. and Ulbrich-Hofmann, R. (1999). Anal. Biochem. 271, 197–199.
Arnold, U., Rücknagel, K.-P., Schierhorn, A., and Ulbrich-Hofmann, R. (1996). Eur. J. Biochem. 237, 862–869.
Arnold, U., Schierhorn, A., and Ulbrich-Hofmann, R. (1998). J. Protein Chem. 17, 397–405.
Brunger, A. T., Brooks, C. L., and Karplus, M. (1985). Proc. Natl. Acad. Sci. USA 82, 8458–8462.
Ciechanover, A. (1998). EMBO J. 17, 7151–7160.
Cohen, F. E. and Prusiner, S. B. (1998). Annu. Rev. Biochem. 67, 793–819.
Creighton, T. E. (1990). Biochem. J. 270, 1–16.
delCardayré, S. B. and Raines, R. T. (1995). J. Mol. Biol. 252, 328–336.
Fan, X.-Y., McPhie, P., and Miles, E. W. (1999). Biochemistry 38, 7881–7890.
Fenton, W. A. and Horwich, A. L. (1997). Protein Sci. 6, 743–760.
Fisher, B. M., Schultz, L. W., and Raines, R. T. (1998). Biochemistry 37, 17386–17401.
Hubbard, S. J. (1998). Biochim. Biophys. Acta 1382, 191–206.
Imoto, T. (1997). Cell. Mol. Life Sci. 53, 215–223.
Janecek, S. (1993). Proc. Biochem. 28, 435–445.
Kasumov, E. A., Volynskaya, A. V., Shishkov, A. V., and Goldanskii, V. I. (1990). Studia Biophys. 136, 167–170.
Klee, W. A. (1965). J. Biol. Chem. 240, 2900–2906.
Kraulis, P. J. (1991). J. Appl. Crystallogr. 24, 946–950.
Liu, W. and Tsou, C.-L. (1987). Biochim. Biophys. Acta 916, 455–464.
Neira, J. L., Sevilla, P., Menéndez, M., Bruix, M., and Rico, M. (1999). J. Mol. Biol. 285, 627–643.
Nogués, M. V., Vilanova, M., and Cuchillo, C. M. (1995). Biochim. Biophys. Acta 1253, 16–24.
Pace, C. N., Laurents, D. V., and Thomson, J. A. (1990). Biochemistry 29, 2564–2572.
Price, N. C. and Johnson, C. M. (1990). In Proteolytic Enzymes–A Practical Approach (Beynon, R. J., and Bond, J. S., eds.), IRL Press, Oxford, 1990, pp. 163–180.
Qi, P. X., Sosnick, T. R., and Englander, S. W. (1998). Nature Struct. Biol. 5, 882–884.
Richards, F. M. and Vithayathil, P. J. (1959). J. Biol. Chem. 270, 17180–17184.
Rico, M., Bruix, M., Santoro, J., Gonzales, C., Neira, J. L., Nieto, J. L., and Herranz, J. (1989). Eur. J. Biochem. 183, 623–638.
Sackett, D. L., Bhattacharyya, B., and Wolff, J. (1994). Biochemistry 33, 12868–12878.
Santoro, M. M. and Bolen, D. W. (1988). Biochemistry 27, 8063–8068.
Schatz, G. (1996). J. Biol. Chem. 271, 31763–31766.
Tsong, T. Y., Hearn, R. P., Wrathal, D. P., Sturtevant, J. M. (1970). Biochemistry 9, 2666–2677.
Tsou, C.-L. (1995). Biochim. Biophys. Acta 1253, 151–162.
Ulbrich-Hofmann, R., Arnold, U., and Mansfeld, J. (1999). J. Mol. Catal. B Enzymatic 286, 125–131.
Wang, L., Chen, R. X., and Kallenbach, N. R. (1998). Proteins 30, 435–441.
Wlodawer, A., Bott, R., and Sjolin, L. (1982). J. Biol. Chem. 257, 1325–1332.
Yang, H.-J. and Tsou, C.-L. (1995). Biochem. J. 305, 379–384.
Yang, H.-J., Li, X. C., Amft, M., and Grotemeyer, J. (1998). Anal. Biochem. 258, 118–126.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Arnold, U., Ulbrich-Hofmann, R. Differences in the Denaturation Behavior of Ribonuclease A Induced by Temperature and Guanidine Hydrochloride. J Protein Chem 19, 345–352 (2000). https://doi.org/10.1023/A:1026479212350
Published:
Issue Date:
DOI: https://doi.org/10.1023/A:1026479212350