Low level formation of potent catalytic IgG fragments mediated by disulfide bond instability.
A highly purified preparation of a monoclonal antibody to vasoactive intestinal peptide (VIP) was analysed by gel filtration. Three peaks of VIP hydrolysing activity were observed, corresponding to the 150 kDa tetramer IgG, 80 kDa dimer of the heavy and light chains (H-L dimer) and 25 kDa L chain monomer. The hydrolytic activity of all three peaks was removed by adsorption on immobilized anti-mouse IgG. The specific activities (CPM hydrolysed/microgram protein) of the H-L dimer and the L chain monomer were more than 30-fold greater than of intact IgG. The presence of small amounts of the antibody fragments in unreduced IgG preparations was confirmed by electrophoresis of overloaded radiolabeled and unlabeled IgG preparations. Increased levels of the fragments were evident after prolonged incubation of a dilute solution of 125I-IgG at 37 degrees C. Iodoacetamide, a sulfhydryl alkylating reagent, suppressed the production of IgG fragments. Incubation of 125I-labeled L chain with unlabeled IgG resulted in incorporation of small amounts of the radioactivity into disulfide bonded 150 kDa IgG tetramer and 50 kDa L chain dimer fractions. These observations implicate disulfide reduction and exchange reactions as the mechanism underlying formation of the IgG fragments. Like the antibody fragments found in unreduced IgG, the L chain monomer and non-covalently associated H-L dimer isolated from reduced and alkylated IgG were capable of hydrolysing VIP. Hydrolysis of VIP by the recombinant L chain subunit was inhibited by excess IgG, suggesting that high affinity VIP binding by IgG limits its hydrolysis by the L chain. These observations suggest that small amounts of high activity antibody fragments may contribute to the catalytic characteristics of the anti-VIP IgG preparation.
Li
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Department of Anesthesiology, University of Nebraska Medical Center, Omaha 68198-6830, USA.
Sun
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M
Gao
Q S
QS
Paul
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S
D000478:Alkylation; D000818:Animals; D017151:Antibodies, Catalytic / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D004220:Disulfides / Q000276:immunology* / Q000494:pharmacology; D004355:Drug Stability; D006868:Hydrolysis; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D007147:Immunoglobulin Light Chains / Q000494:pharmacology; D051379:Mice; D010084:Oxidation-Reduction; D014660:Vasoactive Intestinal Peptide / Q000737:chemistry
D017151:Antibodies, Catalytic; D004220:Disulfides; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D014660:Vasoactive Intestinal Peptide