Antigenic, electrophoretic and binding alterations of human C-reactive protein modified selectively in the absence of calcium.

C-Reactive protein (CRP) is a trace component of normal human serum which has a mol. wt of 105,000 and is composed of five apparently identical subunits arranged in cyclic symmetry. The serum concentration of this protein is known to increase dramatically in response to acute inflammatory or necrotic processes. We report here that in the presence of high concentrations of urea significant antigenic, electrophoretic and binding site modifications of CRP occur selectively in the absence of calcium. CRP treated in this way (designated F-CRP) had a pI of 5.4 and alpha-electrophoretic mobility in contrast to the native molecule which had a pI of 6.4 and gamma-mobility. F-CRP retained a partial antigenic identity to native CRP, displayed decreased intrinsic tryptophan fluorescence, and expressed a new antigenic reactivity. A similar neoantigen was expressed by heating CRP selectively in the absence if calcium (63 degrees C, 5 min). Treatment with guanidinium-HCl or deliberate carbamylation did not produce F-CRP or the expression of the F-antigen. The formation of F-CRP in urea or by heat was prevented by the presence of 0.7 mM or more calcium. CRP treated in this way retained identical characteristics to native CRP. F-CRP chromatographed through Sephadex G-150 in the presence or absence of 6M urea as a protein of apparent mol. wt 75, 000 with no evidence for free CRP subunits. The formation of F-CRP from native CRP resulted in a loss of capacity for calcium-dependent binding to the C-polysaccharide despite the persistence of calcium-independent binding reactivity for polycations. These data suggest that in the presence of sufficient calcium CRP can resist urea- or heat-induced structural denaturation, maintaining antigenic, electrophoretic and binding identity to the native molecule. In the absence of calcium, exposure to urea led to increased electrophoretic mobility and exposure of a new antigenic reactivity, and to alterations in the phosphocholine- but not the polycation-binding sites of the native CRP molecule; this new antigenic reactivity may be of value in further studies on the CRP molecule.

Effect of reduction on the structural, biologic, and immunologic properties of the third component of human complement.

A decrease of the hemolytic function of human C3 results from reduction with sodium borohydride (NaBH4) or with dithiothreitol (DTT) followed by alkylation with iodoacetamide. In the presence of 3.5 x 10(-2) M NaBH4, the loss of hemolytic activity (68%) is paralleled by a loss of C3b binding to EAC14oxy2 cells (67%), immune adherence (71%), and hemagglutinability (74%), but by no loss of C5 convertase function. Sulfhydryl analysis reveals that two disulfide bonds, both presumed to be situated in the C3 alpha subunit, are broken: one at 2.2 x 10(-2) M and one at 3.5 x 10(-2) M NaBH4. A contrasting inactivation profile is observed in the presence of 1.0 x 10(-1) M DTT. Loss of hemolytic function (76%) is paralleled by a decrease of C5 convertase function (87%), whereas C3b binding, immune adherence, and hemagglutinability are either unaffected or not significantly affected. Sulfhydryl analysis indicates the cleavage of four disulfide bonds; three bonds within C3 alpha and beta are broken at 5.0 x 10(-4) M DTT, and one additional bond within C3 alpha at 1.0 x 10(-3) M DTT. Antigenicity as determined by immunoelectrophoresis is unaltered at any of the above concentrations. The implication of these findings is discussed.