Location of the interchain disulfide bonds of the fourth component of human complement (C4): Evidence based on the liberation of fragments secondary to thiol-disulfide interchange reactions

Treatment of human C4 with chemical denaturants and heat produces rapid, autolytic peptide bond cleavage of the α-chain. These α-chain fragments are linked to the parent C4 molecule through disulfide bonds. On more prolonged heating, however, there is liberation of several peptides, including the β-chain, the γ-chain, and a C-terminal α-chain fragment. This reaction is inhibited by iodoacetamide. By using a fluorescent thiol reagent and ¹⁴C-iodoacetamide, the thiol group present on each peptide was analyzed. The results suggest that the thiol residue exposed by cleavage of the thioester bond induces thiol-disulfide interchange reactions to liberate the peptides. Based on the identification of fragments liberated, the kinetics of their appearance, their sulfhydryl content, and the reported primary structure of human C4, a model of the interchain disulfide bonds is proposed in which the amino terminal portion of the α-chain is disulfide-linked to both the β- and γ-chains, whereas the carboxyl terminal portion of the α-chain is disulfide-linked to only the γ-chain.