Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement

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Abstract
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-.alpha.43, thus indicating the presence of only one interchain bond in C3. The two interchain bonds in C4c are .beta.-.alpha.27 nd .alpha.16-.gamma.. The third interchain bond in C4 (.alpha.27-.gamma., tentative) remains to be determined.