Purification and characterization of chicken liver xanthine dehydrogenase.

Previous studies from this laboratory (1) have demonstrated the existence of several different types of “xanthine oxidase” in nature. The enzymes in milk and mammalian tissues appear to be quite similar, but can be differentiated by the inhibitor Antabuse, which affects only the tissue enzyme (2). Antabuse can also be used to separate the dehydrogenase and oxidase activities of tissue xanthine oxidase, inasmuch as it has no effect on the dehydrogenation reaction, but blocks the reoxidation of the enzyme by air. Both of these enzymes are fundamentally different from the xanthine dehydrogenase found in bird tissues, since the latter is not autoxidizable to any significant extent (3). A method was developed for the purification of xanthine dehydrogenase from chicken liver in order to study its properties and the nature of its prosthetic group. It was hoped that a comparison with milk xanthine oxidase would permit an identification of the “dehydrogenase prosthetic group” as separate and distinct from the “oxidase prosthetic group.” Ball (4) originally obtained evidence for the existence of two prosthetic groups in milk xanthine oxidase; one of these was identified as flavin adenine dinucleotide (FAD) and the other was unknown (5). More recent studies (6-8) have demonstrated the presence of molybdenum (8-11) and iron (12) in addition to riboflavin in highly purified preparations of the milk enzyme. The fractionation procedure to be described yielded approximately a 406fold purification of the chicken liver enzyme, and the enzyme so obtained was 97 to 98 per cent homogenous electrophoretically. By any criterion, the purified enzyme was more active than analogous preparations of the milk enzyme. Like milk xanthine oxidase, the chicken liver xanthine dehydrogenase contained riboflavin and molybdenum, but in a ratio of 1: 1 instead of 2: 1. The best preparations of liver enzyme also contained iron, which seemed to be associated with the enzyme, in approximately an 8: 1 ratio with MO. Riboflavin was hardly detectable in the