Effect of reduction and alkylation on structure and function of rabbit IgG antibody--II. Effects on classical pathway C3 convertase formation.
Anaerobic reduction of purified rabbit IgG antibody (Ab) with 1.5 moles of dithiothreitol per mole of Ab at pH 8.0, followed by alkylation, cleaves 39% of the inter-heavy-chain (H-H) disulfide (SS) bonds. This treatment has the following effects on the ability of the Ab to activate the classical pathway of complement. Compared to control Ab, reduced and alkylated (RA) Ab retained 4-5.6% of overall hemolytic activity and 55% of complement-fixing activity at 0 degrees C. Complexes of RA Ab and equivalent amounts of soluble Ag consumed C4, C2 and C3 at 37, 51 and 44%, respectively, of the rate at which these components were consumed by equal concns of complexes containing control Ab. Complexes made with RA Ab bound 18% as much C-1 as those made with native Ab. These data indicate that the principal, if not the only, effect of RA is on C-1 binding. Measurements of the ability of complexes of Ab with cell-bound Ag to bind C-1 showed at most a 20% loss of C-1 binding sites and a ca two-fold decrease in affinity for C-1. Similar results were obtained with purified (activated) C-1 and with native C1 in serum. No significant difference could be detected in the rate of activation of bound C1. Normal rabbit IgG which was reduced and alkylated under the same conditions retained 52% of its H-H SS bonds and 30% of its ability to bind C-1. This finding suggests that the impairment in C-1 binding results from an effect on the C1 binding site itself, rather than from an effect on the ability of the RA Ab to transmit a putative conformational "signal" from the Ag-binding site to the C1 binding site. Finally, our data show that the observed functional effect of reduction and alkylation depends strongly on the assay used to evaluate that effect.
D000478:Alkylation; D000818:Animals; D000936:Antigen-Antibody Complex; D001666:Binding Sites, Antibody; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003167:Complement Activation; D003172:Complement C1 / Q000276:immunology; D050577:Complement C3-C5 Convertases / Q000378:metabolism*; D003168:Complement Fixation Tests; D003171:Complement Pathway, Classical; D006461:Hemolysis / Q000187:drug effects; D007074:Immunoglobulin G / Q000276:immunology*; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D011817:Rabbits
D000936:Antigen-Antibody Complex; D003172:Complement C1; D007074:Immunoglobulin G; D007461:Iodoacetates; D003166:Complement Activating Enzymes; D050577:Complement C3-C5 Convertases