Immunological determination of the order of folding of portions of the molecule during air oxidation of reduced ribonuclease

Abstract

An immunological method was used to follow the folding of different portions of the reduced bovine pancreatic RNase molecule during air oxidation. Antibodies that reacted specifically with segments 1-13, 31-79 and 80-124 of native ribonuclease, as they were folded, were purified by affinity chromatography, using antiserum to native RNase and columns to which the RNase fragments were attached. The kinetics of reaction between these purified antibodies and refolded portions that were produced when reduced RNase was oxidized by air demonstrated the presence of intermediate states of folding, and was consistent with folding of the antigenic determinants in the order 80-124, 1-13 and 31-79. The relative stabilities of each of these segments to thermal denaturation in the native protein provided additional evidence that the native conformation of region 80-124 was a very stable one in the intact molecule. On the basis of these 2 types of evidence, it appears that segment 80-124 contains a nucleation site for the folding of the protein molecule.