Rh antigen immunoreactivity after histidine modification.
125I-anti-D IgG and unlabeled blood group allo-antisera in combination with 125I-protein A were employed in assessing antibody binding to red cells (RBC) treated with histidine reagents. The acylating reagent diethylpyrocarbonate (DEP), the alkylating reagent p-bromophenacyl bromide (pBPB) and the photosensitizer dye Rose Bengal (RB) were used under conditions that usually result in the selective modification of histidine in isolated proteins. Progressive apparent inactivation of the D antigen in ghost membranes occurred with increasing DEP concns, which was not demonstrably reversible by hydroxylamine since this reagent itself inactivated the D antigen. Exposure of red cells to 5 mM p BPB resulted in a 50% decrease in binding of 125I-anti-D IgG. Photo-oxidation of RBC in the presence of Rose Bengal apparently inactivated all the major Rh antigens as detected either by labeled anti-D IgG binding, IgG agglutinating serological reagents, or the binding of 125I-labeled protein A following the sensitization of cells with unlabeled antisera. Under conditions of RB treatment, where hemolysis was absent or minimal, 125I anti-D IgG binding decreased to 38-49% of the level seen in controls. Rose Bengal treatment of R1r RBC revealed varying inactivation of all the Rh antigens, i.e. D 15%, C 89%, c 73%, e 54% inactivated, whereas antibody binding activity of the Fya and Fyb antigens present in the same cell was unaffected. Previous reports as well as the pH profile of anti-D binding have implicated the participation of histidine in Rh antigen expression. Our results are consistent with histidine involvement in Rh activity. Whether Rh antigens have essential histidine(s) involved directly in epitope structure, or instead depend on a critical histidine(s) at the lipid-protein interface that modulates antigen expression remains to be determined.
D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000276:immunology; D006639:Histidine / Q000276:immunology*; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007519:Isoantigens / Q000276:immunology; D010084:Oxidation-Reduction; D012204:Rh-Hr Blood-Group System / Q000276:immunology*; D012395:Rose Bengal / Q000494:pharmacology
D006898:Hydroxylamines; D007519:Isoantigens; D012204:Rh-Hr Blood-Group System; D012395:Rose Bengal; D019811:Hydroxylamine; D006639:Histidine; D004047:Diethyl Pyrocarbonate