Open AccessModification of Protein Properties by Change in Charge
Author(s) -
Shiao Daniel D. F.,
Lumry Rufus,
Rajender Shyamala
Publication year - 1972
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1972.tb01999.x
Subject(s) - succinic anhydride , chemistry , chymotrypsinogen , chymotrypsin , titration , lysine , titration curve , tryptophan , organic chemistry , crystallography , stereochemistry , amino acid , enzyme , biochemistry , trypsin
Succinylated derivatives of bovine chymotrypsinogen A and α‐chymotrypsin were prepared by treatment of the native proteins with succinic anhydride; 60% of the 6‐amino groups of the 13 lysine residues form stable succinylated products. The large negative charge at neutral pH is reflected in electrophoretic and titration behavior but absorption spectra, rotatory‐dispersion spectra and reversible unfolding behavior indicate that the succinylated species are conformationally similar to the parent proteins. Succinylated chymotrypsin is active as an esterase with N ‐acetyl‐ l ‐tryptophan ethyl ester with a small decrease in the maximum‐velocity parameter. The pH dependence suggests that three ionizing groups influence catalytic behavior. The apparent p K a values analyzed in terms of three groups are shifted relative to α‐chymotrypsin. Thermal‐unfolding studies of succinylated chymotrypsinogen demonstrate the presence of sub‐states near pH 7 but is consistent with two‐state behavior at other pH values.