title: Third component of human complement: appearance of a sulfhydryl group following chemical or enzymatic inactivation.
abstract: Treatment of human C3 with hydroxylamine or hydrazine at physiological pH and ionic strength totally abrogates the intrinsic ability of this protein to sustain classical pathway induced hemolysis of sheep red blood cells. Concomitant with the loss of this function the appearance of a single sulfhydryl group can be followed by titration with the sulfhydryl-specific reagents p-(chloromercuri)benzoate, [1-14C]iodoacetamide, 2,2'-dipyridyl disulfide, and 5,5'-dithiobis(2-nitrobenzoic acid). These reagents have also been used to follow the appearance of a free sulfhydryl group on conversion of C3 to C3b with bovine trypsin. Autoradiography of the electrophoretogram of separated alpha-, alpha'-, and beta-polypeptide chains of inactivated, [1-14C]carboxamidomethylated C3 samples has shown that the reactive sulfhydryl group is present in the alpha chain of C3 and in the alpha' chain of C3b, respectively. Digestion of the radiolabeled protein with porcine elastase has localized this sulfhydryl group to a 28 000-dalton fragment of the alpha chain with immunochemical and functional reactivities of the C3d domain. Autoradiographic analysis of a hydrolysate prepared from radioalkylated C3 and subjected to high-voltage paper electrophoresis has shown the labeled amino acid to be [1-14C]-S-(carboxymethyl)cysteine. The susceptibility of native C3 to rapid and irreversible inactivation by nitrogen nucleophiles with the parallel appearance of a cysteinyl residue may indicate the presence of an internal thiol ester. The relationship of the proposed thiol ester to the ability of nascent C3b to acylate cell surface components and carbohydrate polymers is discussed within the context of a transesterification reaction.
mesh_terms: D002729:Chloromercuribenzoates / Q000494:pharmacology; D003176:Complement C3 / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D006898:Hydroxylamines / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
chemical_list: D002729:Chloromercuribenzoates; D003176:Complement C3; D006834:Hydrazines; D006898:Hydroxylamines; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D014357:Trypsin; D007460:Iodoacetamide