The Interaction Between Xanthine Oxidase and Its Antibody*

Abstract

Immunochemical techniques have been applied in many laboratories to investigations of molecular heterogeneity of mammalian enzymes, such as alkaline phosphatases, amylases, lactic dehydrogenases, phosphorylases, and glucose-6-phosphate dehydrogenases. Enzyme-antibody studies may provide information about the enzyme-antibody interaction and specificity, the composition and the structure of enzymes, and the nature of the enzyme-substrate relationship at the active catalytic site.2The present experiments were undertaken to determine the relationship of the antibody binding sites to the active center of the enzyme; to determine the effect of substrate and a competitive inhibitor on the antigenantibody interaction; and to investigate tissue and species enzyme protein immunologic specificity in mature organisms. 4 Materials and MethodsPreparation of xanthine oxidases from various sources. Bovine cream xanthine oxidase (Cr.X.0. ) ( Worthington Biochemical Corporation) was purified by ammonium sulfate fractionation followed by chromatography on hydroxylapatite columns. Purified xanthine oxidase preparations were obtained by similar techniques from homogenates of bovine liver, spleen, and kidney as well as from rat and human liver. Thermally denatured cream xanthine oxidase was prepared by maintaining the enzyme solution at 70" C. for 30 minutes.Production of antibodies. The active and the heat-inactivated Cr.X.0. antigens were injected intradermally as emulsions with complete Freund's adjuvant (Difco) at weekly intervals for four consecutive weeks. A total of 3.3 mg. enzyme protein was given to each animal. In the fifth week, these and control rabbits were bled by cardiac puncture. Purified preparations of y-globulins were isolated from the rabbit sera by the method of Heremans et al. The rabbit y-globulins were then adsorbed with beef serum.