Section 6
Chapter 5,267

Effect of reduction and alkylation on structure and function of rabbit immuno globulin g antibody 2. effects on classical pathway complement c 3 convertase formation

Johnson, B.A.; Hoffmann, L.G.

Molecular Immunology 21(1): 77-88


ISSN/ISBN: 0161-5890
Accession: 005266409

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Anaerobic reduction of purified rabbit IgG antibody (Ab) with 1.5 mol of dithiothreitol/mol Ab at pH 8.0, followed by alkylation, cleaves 39% of the inter-H-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.degree. C. Complexes of RA Ab and equivalent amounts of soluble Ag [antigen] consumed C4 [complement component 4], C2 and C3 at 37, 51 and 44%, respectively, of the rate at which these components were consumed by equal concentrations of complexes containing control Ab. Complexes made with RA Ab bound 18% as much C.hivin.1 as those made with native Ab. Evidently, the principal if not the only effect of RA is on C.hivin.1 binding. Measurements of the ability of complexes of Ab with cell-bound Ag to bind C.hivin.1 showed at most a 20% loss of C.hivin.1 binding sites and .apprx. 2-fold decrease in affinity for C.hivin.1. Similar results were obtained with purified (activated) C.hivin.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.sbd.H SS bonds and 30% of its ability to bind C.hivin.1. The impairment in C1 binding may result 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. The observed functional effect of reduction and alkylation depends strongly on the assay used to evaluate that effect.

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