MECHANISM OF ENZYME INHIBITION BY ANTIBODY - STUDY OF NEUTRALIZATION OF RIBONUCLEASE

Abstract

The degree of inhibition of ribonuclease by univalent antibody (Porter''s fragments) is greater in the presence of substrate of large molecular weight (nucleic acid) than in the presence of substrate of low molecular weight (cyclic cytidylic acid). It follows that inhibitory steric hinderance depends on the relative position of the site of catalysis and of the site of antibody-attachment. The inhibitory properties of fraction I and fraction II obtained from the same pool of antibody were different. The inhibitory potency of the univalent antibody was increased by addition of divalent antibody, directed against univalent antibody. Some univalent fragments were found to give a zone in which the addition of univalent antibody did not lead to noticeable inhibition. With such fragments, it was demonstrated that divalent antibody, directed against univalent antibody, reduced the plateau zone. Divalent antibody directed against univalent antibody could greatly increase the inhibitory capacity of the univalent antibody in univalent antibody excess. This occurred in circumstances in which no insoluble precipitate was formed and in which no lattice formation could occur. It was, therefore, concluded that the inhibitory effect of the divalent antibody would not depend on steric hindrance by a lattice, but on the increase in the firmness of binding between univalent antibody and ribonuclease. It was shown by equilibrium dialysis of antibody-ribonuclease complexes against radioactively labelled competitive inhibitors of ribonuclease that the number of catalytic sites available for combination with competitive inhibitor was similar to the number of catalytic sites which could act on substrate. It was therefore concluded that antibody could only inhibit an enzyme if it blocked access to the catalytic site. It was furthermore demonstrated that two types of antibodies could occur; one combining and inhibiting, the other one combining, not inhibiting and competing with inhibiting antibody.