Spin-labeled ribonuclease A. Effects of chemical, enzymic, and physical modifications on enzyme conformation

Abstract

3-SLHis-105-RNase A [bovine pancreas] is an active derivative of RNase A spin-labeled at the 3 position of the imidazole ring of histidine-105. The spin-labeled enzyme was modified by urea denaturation, reduction, reduction-carboxymethylation, performic acid oxidation and digestion with proteolytic enzymes in order to monitor changes in the geometry of the protein by changes in the EPR spectrum of the nitroxide spin-label probe. The results of these experiments indicate that the spin-label attached to histidine-105 of RNase A is sensitive to modifications affecting the conformational integrity of the molecule and to the reconstituting effects of various active-center ligands.