Abstract
Glycosylation is one of the major naturally occurring covalent modifications of proteins. We have used stem bromelain, a thiol protease with a single, N-glycosylated polypeptide chain as a model to investigate the role of glycosylation of proteins. Periodate oxidation was used to obtain the deglycosylated form of the enzyme. Denaturation studies in the presence of guanidine hydrochloride (Gn·HCl) were performed using fluorescence and circular dichroism spectroscopy. The glycosylated stem bromelain was found to be stabilized by 1.9 kcal/mol as compared to the deglycosylated one. At a given concentration of denaturant, the fraction of denatured protein was higher in the case of deglycosylated stem bromelain. In short, deglycosylated bromelain showed more susceptibility towards guanidine hydrochloride denaturation, indicating the contribution of the carbohydrate part of the glycoprotein to the stability of the enzyme.
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REFERENCES
Lis, H., and Sharon, N. (1993) Eur. J. Biochem., 218, 1-7.
Varki, A. (1993) Glycobiology, 3, 97-130.
Kobata, A. (1992) Eur. J. Biochem., 209, 483-501.
Woods, R. J., Edge, C. J., and Dwek, R. A. (1994) Nat. Struct. Biol., 1, 499-501.
Rudd, P. M., Joao, H. C., Coghill, E., Fiten, P., Saunders, M. R., Opdenakker, G., and Dwek, R. A. (1994) Biochemistry, 33, 17-22.
Joao, H. C., Scragg, I. G., and Dwek, R. A. (1992) FEBS Lett., 307, 343-346.
Joao, H. C., and Dwek, R. A. (1993) Eur. J. Biochem., 218, 239-244.
Lommerse, J. P. M., Kroon Batenburg, L. M. J., Kroon, J., Kamerling, J. P., and Vliegenthart, J. F. G. (1995) J. Biol. NMR, 5, 79-94.
Wormald, M. R., Wooten, E. W., Bazzo, R., Edge, C. J., Feinstein, A., Rademacher, T. W., and Dwek, R. A. (1991) Eur. J. Biochem., 198, 131-154.
Mer, G., Hietter, H., and Lefevre, J. F. (1996) Nat. Struct. Biol., 3, 45-53.
Chu, F. K., Trimble, R. B., and Maley, F. (1978) J. Biol. Chem., 253, 8691-8693.
Schulke, N., and Schmid, F. X. (1988) J. Biol. Chem., 263, 8832-8837.
Vanhoof, G., and Cooreman, W. (1997) Bromelain, in Pharmaceutical Enzymes (Lauwers, A., and Scharpe, S., eds.) Marcel Dekker, Inc., New York, pp. 131-154.
Haq, S. K., Rasheedi, S., and Khan, R. H. (2002) Eur. J. Biochem., 269, 47-52.
Yasuda, Y., Takahashi, N., and Murachi, T. (1970) Biochemistry, 9, 25-32.
Goto, K., Murachi, T., and Takahashi, N. (1976) FEBS Lett., 62, 93-95.
Lenten, L. V., and Ashwell, G. (1971) J. Biol. Chem., 246, 1889-1894.
Laemmli, U. K. (1970) Nature, 227, 680-650.
Arroyo-Reyna, A., Hernandez-Arana, A., and Arreguin-Espinosa, R. (1994) Biochem. J., 300, 107-110.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem., 193, 265-275.
Murachi, T. (1970) Meth. Enzymol., 19, 273-284.
Dubois, M., Gilles, A. K., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Analyt. Chem., 28, 350-356.
Tayyab, S., Siddiqui, M. U., and Ahmad, N. (1995) Biochem. Ed., 23, 162-164.
Siddiqui, S., Hasan, S., and Salahuddin, A. (1995) Arch. Biochem. Biophys., 319, 426-431.
Gascon, S., Neumann, N. P., and Lampen, J. O. (1968) J. Biol. Chem., 243, 1573-1577.
Trimble, R. B., and Maley, F. (1977) J. Biol. Chem., 252, 4409-4412.
Chu, F. K., Watorek, W., and Maley, F. (1983) Arch. Biochem. Biophys., 223, 543-555.
Chu, F. K., Takase, K., Guarino, D., and Maley, F. (1985) Biochemistry, 24, 6125-6132.
Williams, R. S., Trumbly, R. J., MacColl, R., Trimble, R. B., and Maley, F. (1985) J. Biol. Chem., 260, 13334-13341.
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Rasheedi, S., Haq, S.K. & Khan, R.H. Guanidine Hydrochloride Denaturation of Glycosylated and Deglycosylated Stem Bromelain. Biochemistry (Moscow) 68, 1097–1100 (2003). https://doi.org/10.1023/A:1026354527750
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DOI: https://doi.org/10.1023/A:1026354527750