Immobilized Carboxypeptidase A as A Probe for Studying the Thermally Induced Unfolding of Bovine Pancreatic Ribonuclease

Abstract

A method for the preparation of Sephadeximmobilized carboxypeptidase A is presented. This form of the enzyme has the same specific activity as the soluble enzyme at room temperature, but retains its activity at higher temperatures (60-70°). This preparation of immobilized carboxypeptidase A was used, as a proteolytic probe, to investigate the thermally induced unfolding of the C-terminus of ribonuclease A. This technique indicates that the C-terminal residues of ribonuclease A do not unfold until the high-temperature region of the thermal transition (as determined by ultraviolet difference spectrophotometry and optical rotation). © 1975, American Chemical Society. All rights reserved.