Altered antigenic expressions of human C3 and C5, their subunits and fragments following exposure to denaturing conditions

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The present study deals with the antigenic nature of the subunit polypeptide chains of C3 and C5. The purified proteins were reduced and dissociated in 0.01 M dithiothreitol and 6 M guanidine-HCl (GuHCl). Their subunits were separated by filtration over a Biogel A-15 beaded agarose gel bed (width × efficient length = 2.5 × 450 cm) equilibrated with the same reducing-dissociating agents. A comparative analysis using rabbit antisera raised against native C3 or C5 (anti-native C3-C5 antisera) or against the isolated subunits (anti-subunit antisera) gave the following general reaction pattern: anti-subunit antisera formed specific immune precipitates against isolated C3α, C3β and C5β [solubilized in GuHCl or sodium dodecyl sulfate (SDS)] but failed to recognize the native unreduced parent proteins; anti-native C3-C5 antisera showed a reversed reactivity with specificities restricted to the native parent proteins and with no reactivity against the isolated subunits; upon pretreatment with SDS, sodium desoxycholate (DOC) or glycine buffer of a pH of 2.8, unreduced C3 and C5 underwent profound antigenic alterations leading to the full reactivity with the homologous anti-subunit antisera and the concurrent partial loss of reactivity against anti-native C3-C5 antisera. Like the parent protein, various fragments generated from C3 by the ‘aging’ of whole serum at 37°C or by trypsin digestion proved to be susceptible to a similar antigenic transition in the presence of SDS. The subunit origin of these fragments could therefore be identified immunochemically with the aid of anti-C3α-β antisera. The observed immunochemical modification of C3-C5 was apparently effected by detergent binding. Full antigenic transition occurred when C3 was exposed to SDS to yield a maximal binding of approximately 1000 molecules of SDS per molecule of C3, but also under conditions yielding binding at only 5% of this level. The fact that DOC had the same effect as SDS on C3-C5 suggests that these complement proteins possess hydrophobic binding sites in their native state.