Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement.
Disulphide bonds contribute significantly to the maintenance of structural/functional integrity of many proteins. Therefore it was of interest to study the distribution and the effect of disulphides on conformation of complement components C3 and C4. These proteins are precursors of several fragments with various binding sites and distinct physiological functions. The constituents of C3c (beta, alpha 27, alpha 43) and those of C4c (beta, alpha 27, alpha 16, gamma) were investigated, since other fragments of C3 or C4 do not participate in interchain linkages. Inter-and intra-chain disulphide bonds in C3c and C4c were localized by using a modification of conventional SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis such that the change in mobility of disulphide-bond-containing proteins can be detected throughout the transition from a non-reduced to a fully reduced state. Several forms of the alpha 43 fragment from C3, and of the gamma-chain of C4, with different mobilities can exist, depending on the number of intra-chain disulphide bonds reduced. The intermediates (heterodimers) generated by a partial reduction of C3c or C4c were characterized by two-dimensional SDS/polyacrylamide-gel electrophoresis performed in the absence, then in the presence, of beta-mercaptoethanol. The inter-chain linkages in C3c were determined to be beta-alpha 27 and alpha 27- alpha 43, thus indicating the presence of only one interchain bond in C3. The two interchain bonds in C4c are beta-alpha 27 and alpha 16-gamma. The third interchain bond in C4 (alpha 27-gamma, tentative) remains to be determined.
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Janatova
D003176:Complement C3; D015932:Complement C3c; D003181:Complement C4; D015935:Complement C4b; D004220:Disulfides / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology
D003176:Complement C3; D003181:Complement C4; D004220:Disulfides; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D013861:Thiocyanates; C033860:complement C4c; D015932:Complement C3c; D015935:Complement C4b; C008658:2-nitro-5-thiocyanobenzoic acid; D004229:Dithiothreitol