An anti-complementary protein which cross-reacts with antiserum to cobra venom factor (CoF) has been highly purified from Crotalus atrox venom (CAV). On an equal weight basis, the C. atrox factor (CA-F) had one-third the anti-complementary activity of CoF and similarly consumed the terminal components of complement via the alternative pathway. The subunit compositions of CoF and CAV were similar, each being composed of alpha and beta chains held together by covalent and non-covalent bonds. Agarose gel diffusion analysis using monospecific antiserum to CoF detected a reaction of partial immunological identity between CoF and CA-F and between C3 and CA-F with the CoF and C3 precipitin lines spurring over the CA-F lines. Thus CA-F probably represents a C. atrox C3 protein which is capable of activating the alternative complement pathway via the amplification loop in a manner analogous to that of C3b and CoF.

Four anti-complementary proteins with distinct physicochemical and immunochemical properties have been highly purified from Crotalus atrox venom. These factors are extremely heat labile and are composed of single polypeptide chains. The isolated factors inactivated complement components by proteolytic cleavage and they exhibited differences in their potency and specificity to their complement substrates.

The kinetics of cleavage of C3 by trypsin was analyzed by electrophoresis in agarose and in polyacrylamide gels containing sodium dodecyl sulfate and the data obtained were used to construct an anatomical model for C3 showing the sites of tryptic attack, the fragments generated, and their composition. Trypsin was shown to cleave C3 in a stepwise fashion. The attack was initially directed at the alpha-polypeptide chain and resulted in the generation of C3a and C3b. Further cleavage of the alpha-chain of C3b, converted it into C3b1 and then into C3d and C3c. Cleavage of the beta-chain by trypsin occurred only at the C3c stage with the release of a small peptide (m.w. 12,000) from C3c and the formation of C3c'. On immunoelectrophoresis, C3c' had a less anodal mobility compared to the beta1A mobility of C3c. C3a, once formed could be further cleaved to give residual fragments with decreasing net positive charge. Exposure of C3 to acid conditions, pH 5.0 or below, rendered the molecule exceedingly susceptible to tryptic degradation.