The Effect of Specific Antibodies on Xanthine Oxidase from Various Sources

Abstract

Purified rabbit antibodies were prepared against native and thermally denatured bovine cream xanthine oxidase (Cr.X.O.). Each antibody reacted with lines of identity to both enzymes when tested in the Ouchterlony plate. Similar cross-reactions were demonstrated in liquid precipitin tests with concomitant inhibition of enzyme activity by either antibody. Neither the substrate, xanthine, nor the competitive inhibitor, 4-amino-6-hydroxypyrazolo-(3,4-d)pyrimidine, both of which combine with the active center of the enzyme, prevented the antigen-antibody interaction. Thus, physical or chemical modifications of the active center of the enzyme do not influence its immunologic specificity, indicating that antibodies are not formed primarily to the active center of the enzyme. The retention of considerable catalytic activity by the enzyme-antibody precipitate indicates that steric blockade of the active center by proximal binding of the antibodies is incomplete. A technique for continuously measuring the rate of antigen-antibody precipitation and residual enzyme activity has been described. At equivalence, the rate of precipitation was most rapid and the catalysis of xanthine by bovine Cr.X.O. was maximally reduced, with residual enzyme activity localized mainly in the precipitate. Species specificity was shown by the absence of interaction between antibody to bovine Cr.X.O. and rat liver xanthine oxidase. These antibodies, however, did cross-react with xanthine oxidase isolated from various bovine tissues and from human liver.