THE FUNCTIONS OF THE 9TH COMPONENT OF HUMAN-COMPLEMENT ARE SUSTAINED BY DISULFIDE BONDS WITH DIFFERENT SUSCEPTIBILITIES TO REDUCTION

Purified C9 with expected hemolytic and polymerizing activities was fo und to contain approximate to 0.2 mol of sulfhydryl groups/mol of C9. By proteolysis of C9 with labeled SH groups, the SH residues on intact C9 were mapped to Cys-359 and Cys-384 which, presumably, form an intr a-domain disulfide bond in the intact molecule. The blocking of these sulfhydryl residues by alkylation, however, had minimal influence on t he functions of C9. On the other hand, reduction of C9 by 1 mM dithiot hreitol (DTT) (6-fold molar excess over Cys residues) followed by alky lation resulted in a complete block of polymerization activity and a 5 0% loss of C9 hemolytic activity. In contrast, the ability of C9 to bi nd EAC1-8 remained largely unaffected. The loss of poly-C9 formation a ctivity correlated with the alkylation of approx. 6 liberated sulfhydr yl groups. Hemolytic activity was abolished by treatment with > 5 mM D TT which allowed the liberation of approximate to 18 sulfhydryl groups . Most of the DTT-susceptible disulfides were within the C9a fragment (an N-terminal peptide derived by thrombin). Thus, three major functio ns of C9, EAC1-8 binding, polymerization, and hemolytic activity, are sustained by disulfide bond-dependent conformational motifs with diffe rent susceptibility to reducing reagents. The maintenance of the N-ter minal C9a region is essential for polymerization, but not EAC1-8 bindi ng activity of C9. Taken together, the results of the present study di fferentiate in molecular terms several of the functional portions of C 9, and stress the significance of intra-chain disulfide linkages in ma intaining the structural components necessary for the functions of C9.