Cleavage of Human C5 by Trypsin: Characterization of the Digestion Products by Gel Electrophoresis

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Abstract
Human C5 is composed of two nonidentical polypeptide chains, α and β (m.w. 130,000 and 80,000, respectively) linked together by disulfide bonds and noncovalent forces. Cleavage of C5 by trypsin was restricted to the α-chain and resulted in the generation of major fragments with increased anodic mobilities. Limited digestion of C5 by trypsin (substrate to enzyme ratio 10:1 w/w at 37°C for 1 min) resulted in the release of a small terminal α-chain peptide (α1, m.w. 15,000) probably analogous to C5a, from a large fragment, C5b (m.w. 195,000) composed of an intact β-chain disulfide linked to an α-chain that has a lower m.w. (α′ 115,000). Further digestion (37°C, 5 min) resulted in cleavage of the α-chain at multiple sites with the production of three peptides from the α′-chain (α2I, 23,500; α2II 15,700 and α2III 10,200) and a residual fragment, C5c (m.w. 144,000). The α1 and α2 peptides are not covalently linked to the β-chain nor to one another. The C5c fragment on the other hand is composed of small peptides of the α′c chain (α3 14,000; α4I 9,000; α4II 11,000; α5 23,000 to 30,000) which are linked to the β-chain and also probably to one another by covalent bonds. Secondary cleavage occurred upon prolonged digestion with trypsin (37°C, 20 min), and this resulted in the progressive erosion of the α′c peptides and the conversion of C5c to smaller C5c-like species.