Highly purified human C5 was subjected to limited trypsin digestion (0.42 mol affinity-purified trypsin/mol C5) at pH 7.4 and 37°C. Samples were removed after various time intervals, treated with phenylmethylsulfonyl fluoride to inhibit trypsin activity, and were analyzed by SDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) in the presence or absence of β-mercaptoethanol (βME). The first trypsin-mediated cleavage event was located in the C5α chain and resulted in the production of a 25,000 ± 3000 m.w. (α5) polypeptide and a 90,000 ± 3000 m.w. (α1) fragment. The larger C5α chain fragment, C5α1, produced by trypsin clearly expressed a smaller m.w. than the C5bα chain (102,000 ± 3000 m.w.) produced by the C5 convertase enzymes of either complement activation pathway. The second site of trypsin action resulted in the hydrolysis of the C5α1 chain into C5α2 (58,000 ± 3000 m.w.) and C5α4 (32,000 ± 3000 m.w.) fragments. The C5α2 polypeptide chain was subsequently degraded to a C5α3 fragment (44,000 ± 3000 m.w.). Under the conditions employed in these experiments, only the C5α chain subunit was susceptible to trypsin digestion; the β-subunit was completely resistant. Two-dimensional SDS-PAGE analysis of trypsin-digested C5 (the first dimension was conducted under nonreducing conditions and the second dimension was performed in the presence of βME) demonstrated that all of the C5 fragments produced by the first three trypsin cleavage events (α1 through α5) were disulfide-bonded together, thereby generating a series of trypsin-modified C5 molecules (C5’, C5”, C5’“) possessing the same apparent m.w. as native C5 (210,000 ± 5000). Collectively, these results indicate that limited trypsin digestion of human C5 did not result in the production of C5a and C5b fragments. Nevertheless, limited trypsin digestion did result in the activation of human C5 to induce human neutrophil chemotactic and lysosomal enzyme-releasing activities. Detailed correlation experiments demonstrated that the first trypsin-mediated C5α chain cleavage event, i.e., conversion of C5α to C5α1 and C5α5 polypeptide fragments, resulted in the expression of C5a-like biologic activities by trypsin-modified C5.
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Wetsel et al. (1982) studied this question.