| Description |
The third component of guinea pig complement (C3) was isolated from EDTA-plasma by poly(ethylene)glycol precipication, depletion of plasminogen on Sepharose-L-lysine, chromatography on DEAE-Sephacel, and gel filtration on Sepharose CL-6B. IgG was removed in a final step with Protein A-Sepharose. The recovery of functional activity was 56% with a purification of 81-fold. SDS-PAGE indicated an a3 chain structure. The apparent molecular weights of the alpha- and beta-3-chains were 115,000 ± 12,000 and 65,000 ± 7,000, respectively. When compared with human C3, the alpha-chain was of similar size, but, the guinea pig beta-chain was significantly smaller. This difference may in part be due to carbohydrate since guinea pig beta-chain, unlike human beta-chain, failed to stain for carbohydrate following periodic acid oxidation. The alpha- and beta-chains were separated on a column of Sepharose CL-4B equilibrated in 0.2% SDS and the amino-terminal structure of each was determined by Edman degradation. The structure of each chain was highly concordant with the corresponding chain of human C3. Incubation of guinea pig C3 with (14C) methylamine resulted in a final uptake of 0.76 moles/mole into the alpha-chain with the simultaneous acquisition of a free sulfhydryl group (SH). Human and guinea pig (14C) methylamine-inactivated C3 were immobilized on activated Thiol-Sepharose and digested with porcine elastase. SDS-PAGE of bound material released with L-cysteine revealed a primary band with a molecular weight of 33,000 (C3d) which retained the (14C) label. Amino-terminal structure of human and guinea pig C3d indicated 92% homology for the 29 residues identified. The (14C) label was released with a Glx residue at step 26 and the SH group, which appears upon nucleophilic inactivation, was released with cysteinyl residue at step 23. This indicates that a thiolester site is located at this position in the amino-terminal region of C3d. For human, a 35 residue tryptic peptide containing the thiolester site was previously isolated and the positions 1-16 reported (Tack et al., 1980, PNAS 77: 5764). The complete sequence of the 35 residue peptide is reported here and commences with the histidyl residue at position 15 in C3d. |
