Effects of acetylation and guanidination on alkaline conformations of chymotrypsin

Antonio T. Fojo, Philip L. Whitney, William M. Awad

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12 Scopus citations

Abstract

Guanidination leads to stabilization of several globular proteins, including bovine chymotrypsinogen, as determined by hydrogen isotope exchange (P. Cupo, W. El-Deiry, P. L. Whitney, and W. M. Awad, Jr. (1980) J. Biol. Chem. 255,10828-10833). The present study examined the binding of proflavin to guanidinated, acetylated, and native chymotrypsins in order to compare conformational flexibilities. The order of decreasing alkaline stabilities of the catalytically active conformations of the different δ-chymotrypsin forms was guanidinated, native, and acetylated proteins; δ-chymotrypsin showed greater stability than α-chymotrypsin. In each case removal of calcium reduced the amount of the catalytically active conformation. The alkaline pH dependence for the decrease of the catalytically active conformation of guanidinated α-chymotrypsin could not be attributed to the titration of a single group, indicating that the α-amino group of isoleucine-16 is not the sole feature regulating the conformational transition for this derivative. At neutral pH values δ-chymotrypsin exists completely in an active conformation and the percentage of α-chymotrypsin in this form is only slightly lower. These differences from earlier results are possibly due to differences in buffers, calcium ion concentrations, and ionic strength. The rate of inactivation of guanidinated δ-chymotrypsin with methyl acetimidate was much lower than the corresponding rate for the native enzyme. This suggests that guanidination increases enzyme stability which in turn leads to a reduced accessibility of the α-amino group of isoleucine-16.

Original languageEnglish (US)
Pages (from-to)636-642
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume224
Issue number2
DOIs
StatePublished - Jul 15 1983

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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