Cell-bound C4b resists reduction by reducing agents: analysis by chain structure and by hemolytic activity

EA3H-C4hu treated with 2-mercaptoethanol (MSH) or dithiothreitol (DTT) were analyzed for residual chains and hemolytic activity. MSH treatment resulted in no loss of the chains of C4b and no loss of reactivity with a polyclonal anti-human C4 rabbit antibody, and the cells did not lose their ability to generate SAC4b2a. DTT-treated cells lost about 70% of the gamma-chain and over 95% of C4b activity; there was no loss of alpha' and beta chains or the ability to react with anti-C4 antibody. Kinetic studies indicated that the remaining 30% of the gamma-chains could not be removed by prolonged incubation with DTT, implying heterogeneity of cell bound C4b. The data also imply that the gamma-chain is important in the generation of SAC4b2a.