Abstract
The primary structure around the single cysteinyl residue of chicken pepsin was investigated by binding the protein via this residue to an insoluble carrier. Carriers stable towards reagents used for the fragmentation of proteins and sequence analysis were prepared by coupling a spacer arm to polyN-hydroxymethyl acrylamide using a thioether bond that is potentially cleavable by mercuric ions (1). Phenacyl bromide group, attached to the free end of the spacer, reacted rapidly and specifically with the cysteinyl residue of chicken pepsin. Up to 300 mg of the enzyme were bound to 1 g of carrier.
The polymer-bound protein was cleaved by trypsin or by cyanogen bromide or by a sequence of both. Fragments of 40–120 amino acid residues, depending on the method of cleavage, remained attached to the polymer through the cysteinyl residue. The compositions and partial sequences of these fragments revealed that the cysteinyl residue is located within or in the vicinity of a loop in the molecule formed by a disulfide bond.
Similar content being viewed by others
References
Veber, D. F., Milkowski, J. D., Varga, S. L., Denkewalter, R. G., and Hirschmann, R. (1972),J. Am. Chem. Soc. 94, 5456.
Bohak, Z. (1969),J. Biol. Chem. 224, 4638.
Becker, R., Shechter, Y., and Bohak, Z. (1973),FEBS Lett. 36, 49.
Brocklehurst, K., Carlsson, J., Kierstan, M. P. J., and Crook, E. M. (1974),Methods Enzymol. 34, 531.
Egorov, T. A., Svenson, A., Ryden, L., and Carlsson, J. (1973),Proc. Natl. Acad. Sci. USA 72, 3029.
White, C. A., and Kennedy, J. F. (1980),Enz. Microb. Technol. 2, 83.
Gabison, D., M. S. Thesis (1973), The Faculty of Agriculture, The Hebrew University of Jerusalem.
Bohak, Z., and Li, S. L. (1976),Biochem. Biophys. Acta 427, 153.
Friesell, R. W., and Mackenzie, C. G. (1958),Methods of Biochemical Analysis, Glick, D., ed., Vol. 6, p. 63.
Kamogowa, H., and Sekiya, T. (1961),J. Poultry Sci.,50, 122.
Inman, J. K. (1974),Methods Enymol. 34, 30.
Raadsveld, C. W. (1935),Recu. Trav. Chim. Pays-Bas 54, 813.
Gross, E. (1967), inMethods Enzymol. 11, 238.
Ambler, R. P. (1972), inMethods Enzymol. 25, 143.
Laursen, R. A., Bonner, A. G., and Horn, M. J. (1975), inInstrumentation in Amino Acid Sequence Analysis, Perham, R. N., ed., Academic Press, New York, p. 73.
Bricker, C. E., and Johnson, H. R. (1945),Ind. Eng. Chem. Anal. Ed. 17, 400.
Atherton, E., and Sheppard, R. C. (1974), inPeptides, Wolman, Y. (ed.), Proc. of the 13th European Peptide Symposium, Kiryat Anavim, Israel, p. 123.
Moroder, L., Marchiori, F., Borin, G., and Scoffone, E. (1973),Biopolymers 12, 493.
Einhorn, A. (1905),Justus Liebigs Ann. Chem. 343, 265.
Goldstein, L., and Manecke, G. (1976), inImmobilized Enzyme Principles. Wingard, L. B., Katchalski-Katzir, E., and Goldsein, L., Eds. Academic Press, New York, Vol. 1, p. 23.
De-Jong, J. I., and De-Jonge, J. (1952)Recu. Trav. Chim. Pays-Bas 71, 643.
Hartley, B. S. (1970)Biochem. J. 119, 805.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Amarant, T., Bohak, Z. Immobilization of proteins as a tool for studying primary structure around their cysteinyl residues. Appl Biochem Biotechnol 6, 237–250 (1981). https://doi.org/10.1007/BF02780801
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02780801