LOCATION OF THE ANTIGENIC DETERMINANTS OF BOVINE PANCREATIC RIBONUCLEASE

Four antigenic regions of native bovine pancreatic RNase were located by using antibodies that react specifically with segments 1-13, 31-79 and 80-124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicates that there are at least 3 antigenic determinants to which antibody molecules can bind simultaneously in the presence of excess antibodies. Analysis of binding data for each purified specific antibody preparation, carried out by the method of Berzofsky et al., leads to an estimate of 4 for the number of antigenic determinants in RNase; this estimate had also been made earlier by Stelos et al. One determinant is associated with each of segments 1-13 and 80-124 and 2 with segment 31-79. No antigenic activity could be detected for segment 14-29 either in native ribonuclease or in the free fragment. These conclusions are based on: the use of specific peptides to isolate purified antibodies by affinity chromatography, immunoprecipitation of an antigenic peptide from the peptic digest of RNase, competitive inhibition studies with various peptide and protein fragments [cyanogen bromide fragments 1-13, 31-79 and 80-124, the tryptic peptides 40-61 and 105-124, S-peptide, S-protein and des(121-124)-RNase], and comparison and evaluation of the published effects on antigenicity of chemical and enzymatic modifications and changes in sequence among homologous ribonucleases. These approaches provide evidence that the 4 antigenic determinants are localized around the .alpha.-helical portion of segment 1-10, somewhere in segment 40-61, at the .beta. bend in segment 63-75, and either at the .beta. bend or .beta. sheet in segment 87-104 of native RNase.