The use of protein A and concanavalin A to examine the possible role of the carbohydrate of IgG in the binding of Clq

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The suggestion that Fc-carbohydrate of rabbit IgG becomes accessible to conA on cross-linking by Staphylococcal protein A and can play a role in fixing complement (Langone et al., 1978 a) was examined specifically for its relevance to Clq binding. On chromatography of pooled rabbit IgG on conA-Sepharose, 80 ± 5% of IgG shows no interaction with conA—Sepharose. Five per cent of IgG is bound and eluted with 0.1 M 1- O-methyl-α- d-glucopyranoside. The apparent K d of the unretarded fraction for conA was less than 140 μ M, whereas that of pooled IgG was 14 μ M, as measured by competition for 125I-labelled conA with guinea pig erythrocytes. No change in the K d of the unretarded fraction of IgG for conA could be detected in the presence of protein A. A change of up to two-fold was observed in pooled IgG, and must be a result of heterogeneity of the carbohydrate in either the Fab or the Fc region of IgG. The fraction of IgG which did not bind conA showed the same Clq-binding activity as pooled IgG in 125I-labelled Clq binding assays, so that there can be no direct relation between conA-binding activity and Clq binding. The binding of Clq to IgG was not affected by concentrations of conA (1mg/ml) and human transferrin glycopeptide (2 mM) under conditions where inhibition would be expected if complete exposure of the Fc-carbohydrate to solution was important for binding Clq.