Red cell surface cysteine residue (285) of D polypeptide is not essential for D antigenicity.

Abstract

Only one surface cysteine residue (285) has been thought to be involved in D antigenicity, according to studies using lyophilized or nonlyophilized red cell membranes. However, it has been reported that a 17-kDa chymotryptic fragment containing the N-terminus but not this cysteine residue is associated with D antigenicity. The role of the sulfhydryl (SH) group in D, c, and E antigenicity is assessed by using intact red cells treated with the reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and 2-(4'-maleimidylanilino)- naphthalene-6-sulfonic acid. Antigenicity was appraised by hemagglutination titers and immunoprecipitation using human anti-D, -c, and -E. Treatment with N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid) at various concentrations (< or = 5 mM) or for various times (< or = 120 min) did not cause significant decrease in hemagglutination titers as compared to untreated intact red cells. Moreover, immunoprecipitation of Rh antigen-carrying peptides by human anti-D was not affected by prior treatment with N-ethylmaleimide or 2-(4'maleimidylanilino)-naphthalene-6-sulfonic acid. Efficacy of blockage of SH groups was demonstrated by inhibition of palmitic acid uptake by the Rh polypeptides for prior treatment with N-ethylmaleimide and by the presence of fluorescent Rh polypeptides for prior treatment with 2-(4'maleimidylanilino)-naphthalene-6-sulfonic acid. SH group involvement is not essential for D, c, or E antigenic expression in intact red cells.