3RD COMPONENT OF HUMAN-COMPLEMENT - APPEARANCE OF A SULFHYDRYL-GROUP FOLLOWING CHEMICAL OR ENZYMATIC INACTIVATION

Abstract

Treatment of human C3 [complement component 3] 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 SH group can be followed by titration with the sulfhydryl-specific reagents p-(chloromercuri)benzoate, [1-14C]iodoacetamide, 2,2''-dipyridyl disulfide and 5,5''-dithio-bis(2-nitrobenzoic acid). These reagents were also used to follow the appearance of a free SH 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 showed that the reactive SH 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 localized this SH 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 showed the labeled amino acid to be [1-14C]-S-(carboxymethyl) cysteine. The susceptibility of native C3 to rapid and irreversible inactivation by N 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.