Sequence determination of the thiolester site of the fourth component of human complement.

The fourth component of complement (C4) is inactivated by treatment with methylamine. This property is shared wit the third component (C3) and with alpha 2-macroglobulin. In each instance, the reaction with methylamine is stoichiometric, covalent, and accompanied by the appearance of a thiol group. These data are consistent with the presence of an internal thiolester bond. Incubation of C4 with [14C] methylamine in the presence of activated thiol-Sepharose resulted in immobilization of the protein via its active-site thiol. Analysis of bound C4 indicated incorporation of 1.12 mol of [14C]methylamine per mol of protein. Digestion of the immobilized protein with porcine elastase resulted in the release of C4 beta- and gamma-chains and lower molecular weight fragments. The 14C label, however, was retained on the Sepharose beads. Subsequent release of bound material with L-cysteine indicated that the radiolabel was associated with two polypeptides of Mr 25,000 [C4d(ela25)]. The released material was dialyzed and the active-site thiol was radioalkylated with iodo[2-3H]acetic acid. C4d(ela25) was further purified by chromatography on Sephadex G-100 and, after reduction and alkylation, on Sepharose CL-6B in 0.2% NaDodSO4. The C4d(ela25) pool, containing 0.83 mol of [14C]methylamine per mol of iodo[2-3H]acetic acid, was subjected to automated sequence analysis. S-carboxy-[3H]methylcysteine was released at step 21 and gamma-glutamyl-[14C]methylamide was released at step 24. The recovery of radiolabel at positions 21 and 24 confirmed the originally calculated 14C/3H incorporation ratio and further indicated that the radiolabels were present at single sites in the C4 molecule. Comparison of the derived primary structure for the thiolester site in C4 with those for the corresponding regions in C3 and alpha 2-macroglobulin has shown sequence identity. Further comparisons among these three proteins have indicated additional homologies on both the NH2- and COOH-terminal sides of the thiolester site.