Immunochemistry of sperm-whale myoglobin—XXIII: investigation of the independence of the five antigenic reactive regions by immunoabsorbent studies

Abstract

Extensive chemical and immunochemical work in our laboratories has now culminated in the accurate delineation of the entire antigenic structure of myoglobin (Mb). These studies have all been reported. Five antigenic reactive regions have been accurately narrowed down, and were six or seven residues in size. In the present work, antibodies against MbX were prepared from two antisera, labelled with I 125 and adsorbed on Mb-Sepharose immunoabsorbent. The five antigenic reactive regions, prepared by organic synthesis, were each studied with regard to their abilities to displace specific antibodies from the Sepharose-Mb-antibody complex. Batchwise displacement with one reactive region only, followed by recovery of total elutable antibody, showed that of the five antigenic reactive regions only region 56–62 failed to elute specific antibodies in this fashion. The other four reactive regions each effected appreciable displacement of antibody, the extent of which depended on on the immunochemical reactivity of that region previously reported from solution immunochemistry. Lack of reaction of region 56–62 was attributed to steric hindrance on the immunoabsorbent, exerted by antibody molecules bound to the other reactive regions. The obstruction prevented approach of free region 56–62 to the corresponding antigen-antibody binding sites. This was confirmed by serial displacement studies with the five synthetic peptides. Removal in series of antibody molecules bound to the other four reactive regions of Mb first, followed by elution with peptide 56–62 enabled the displacement of specific antibodies by the latter peptide. The amount of displacement with a given immunoabsorbent agreed well with the expected reactivity of the region with that antiserum. Serial elution with five synthetic regions usually displaced about 81% of the total elutable antibody. Reabsorption of specific antibody, thus eluted by a giben reactive region, on Mb-Sepharose followed by serial elution with the five antigenic reactive regions showed that this antibody was displaced only by that particular reactive region. It was therefore concluded that in early course antisera, the five antigeniv reactive regions of Mb were immunochemically independent and showed no immunochemical interaction.