Effect of temperature upon the conformations of carboxypeptidase A (Anson), A γLeu, A γVal, and A α+β

Abstract

1. 1. The conformations of several bovine carboxypeptidase A (peptidyl- l-amino acid hydrolase, EC 3.4.2.1) preparations were compared using the circular dichroism (CD) spectra from 200 to 330 nm. Carboxypeptidase A γ Val, A γ Leu and A α+β (70% A α Leu and 30% A β Val) have nearly identical CD spectra. After considering the origin of the various CD bands, it is suggested that these species have similar backbone conformations and similar environments for their tryptophanyl side chains. 2. 2. The CD spectrum of carboxypeptidase A (Anson) differes in one respect from that of the homogeneous species (A γ Val and A γ Leu). In the Anson preparation the broad, positive CD band between 255 and 270 nm is about twice as intense as that in either carboxypeptidase A γ Val or A γ Leu. The intensification of this CD band in the Anson preparation may possibly be due to an impurity. Alternatively the conformation of the carboxypeptidase A γ molecules may be altered during their isolation from the Anson preparation. 3. 3. The effect of temperature upon the conformations of carboxypeptidase A γ Val, A γ Leu, A α+β and A (Anson) was examined by recording CD spectra between 24 and −196°. The far ultraviolet CD bands of these carboxypeptidase preparations are not altered by cooling, which suggests that the peptide backbone conformation is relatively rigid. In contrast, the CD fine structure arising from the tryptophanyl side chains is greatly intensified upon cooling the various preparations. This behavior results because the tryptophanyl side chains of carboxypeptidase A exist in multiple conformations at 24°. Cooling shifts more side chains into the conformer having the lowest energy. Apparently some side chains of carboxypeptidase A possess appreciable motility at room temperature.