LOW-LEVEL FORMATION OF POTENT CATALYTIC IGG FRAGMENTS MEDIATED BY DISULFIDE BOND-INSTABILITY

A highly purified preparation of a monoclonal antibody to vasoactive i ntestinal 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) a nd 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/mu g protein) of the H-L dimer and the L ch ain monomer were more than 30-fold greater than of intact IgG. The pre sence of small amounts of the antibody fragments in unreduced IgG prep arations was confirmed by electrophoresis of overloaded radiolabeled a nd unlabeled IgG preparations. Increased levels of the fragments were evident after prolonged incubation of a dilute solution of I-125-IgG a t 37 degrees C. Iodoacetamide, a sulfahydryl alkylating reagent, suppr essed the production of IgG fragments. Incubation of I-125-labeled L c hain with unlabeled IgG resulted in incorporation of small amounts of the radioactivity into disulfide bonded 150 kDa IgG tetramer and 50 kD a L chain dimer fractions. These observations implicate disulfide redu ction 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. Hydrol ysis of VIP by the recombinant L chain subunit was inhibited by excess IgG, suggesting that high affinity VIP binding by IgG limits its hydr olysis by the L chain. These observations suggest that small amounts o f high activity antibody fragments may contribute to the catalytic cha racteristics of the anti-VIP IgG preparation. Copyright (C) 1996 Elsev ier Science Ltd.