The mode of attenuation of erythrocyte membrane Rh0(D) antigen activity by 5,5'-dithiobis-(2-nitrobenzoic acid) and protection against loss of activity by bound anti-Rh0(D) antibody.
The Rh0(D) antigen activity of human erythrocyte membranes is lost on treatment with the aromatic disulfide, 5,5'-dithiobis-(2-nitrobenzoic acid), reacting by specific disulfide interchange with membrane thiols. The inactivation rate of the antigen was first order with respect to inhibitor concentration and a double reciprocal plot of the inactivation rate constants against the sulfhydryl reagent concentration was linear, giving an apparent dissociation constant of 1.25 mM and indicating that a binding step preceded covalent interaction. In a study of the binding characteristics of anti-Rh0(D) to the 2-nitro-5-thiobenzoate derivative of the membrane Rh0(D) antigen, the maximum number of binding sites fell with increasing sulfhydryl reagent concentration but the apparent association constant also diminished at the same rate. The inactivation of the Rh0(D) antigen was greatly retarded by the presence of bound anti-Rh0(D), a protective effect not seen with normal serum. Bound anti-Rh0(D) antibody protects against both the loss of sites and the change in affinity brought about by DTNB. It is concluded that a single critical thiol whose reactivity is altered by bound anti-Rh0(D) antibody is involved in the membrane Rh0(D) antigenic determinant site. These observations may permit extensive probing of the microenvironment of this essential cysteine residue.
D004228:Dithionitrobenzoic Acid / Q000378:metabolism / Q000494:pharmacology*; D004306:Dose-Response Relationship, Immunologic; D004910:Erythrocyte Membrane / Q000276:immunology*; D004912:Erythrocytes / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000276:immunology*; D009579:Nitrobenzoates / Q000494:pharmacology*; D012204:Rh-Hr Blood-Group System / Q000276:immunology*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors
D007518:Isoantibodies; D009579:Nitrobenzoates; C033242:RH-antibodies; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid