Electrophoretic and 31P nuclear magnetic resonance evidence for alterations in conformation and net charge on phosphorylation and “aging” of α-chymotrypsin

Abstract

Native α-chymotrypsin and its phosphoenzyme and “aged” phosphoenzyme derivatives are conveniently differentiated by polyacrylamide gradient gel electrophoresis in a pH 4.5 buffer. The changes in relative mobilities most likely reflect alterations in conformation induced by phosphorylation and, for the aged product, also the formation of the negatively charged substituent. Similar electrophoretic separations are achieved for the corresponding derivatives of γ-chymotrypsin and α-chymotrypsinogen A, but not for those of trypsin. Aged α-chymotrypsins give rise to two 31P resonances, also suggesting conformational isomerization of the enzyme, and the chemical shifts exhibit a small, reversible, pH dependence. A third phosphoenzyme arises on longer term incubations at pH 7.6. Under the conditions examined, 31P n.m.r. spectroscopy does not allow differentiation of the aged phosphoenzymes, intermediates in their formation, or the phosphorylated but non-aged analogs.