The isolation and characterisation of bovine C4a
C4a, a peptide released on activation of complement component C4 by Cls, was isolated directly from bovine plasma. A yield of 27% or 5.14 mgl⁻¹ was obtained. The complete primary sequence of bovine C4a was determined and compared with those of C3a and C5a, anaphylatoxins which are similarly released on activation of complement components C3 and C5 by C3 and C5 convertase. Gross similarity was observed between bovine C4a and C3a and C5a from a number of species. Six cysteine residues were conserved, four as cysteinylcysteine sequences and two as single cysteines. Also notable was the high content of basic amino acids which accounted for the molecules net positive charge on pH 4.3 gel electrophoresis and pH 8.6 cellulose acetate electrophoresis. The absence of tryptophan explained the molecules low absorbance at 280 nm. This, coupled with its low molecular weight, 8968, made C4a difficult to detect during purification. Recent sequence data on human C4a indicated 83% identitiy of 16% residue replacements between the bovine and human C4a sequences. Bovine C4a appears to be almost equally identical to human and porcine C3a having 33.76% and 37.60% identity respectively. Similarly the bovine sequence has 38.90% and 35.00% identity respectively with those of human and porcine C5a. A functionally important carboxy-terminal arginine is conserved between all three anaphylatoxins as is the Leu at residue 75 in C3a and bovine C4a and residue 72 in C5a. Bovine C4a and human and porcine C3a are identical in the carboxy-terminal residues 74-77. Studies by Hugli and Erickson (1977) using synthetic peptide analogues of this region suggest that bovine C4a will have a greater biological activity than human C4a, the Ala at residue 73 in human C4a being replaced by Val in bovine C4a. The sequence results obtained indicate that C4a is indeed the third anaphylatoxin of the complement system and support the theory that the parent molecules C3, C4 and C5 are the product of primordial gene duplication.