220] d-Amino acid oxidase and its complexes (hog kidney)

Abstract

Publisher Summary This chapter focuses on D-Amino Acid Oxidase and Its Complexes. The enzyme activity has been assayed by measuring, manometrically or polarographically, the rate of oxygen consumption because of the oxidation of substrate by this enzyme. Using benzoate as a stabilizer during the purification procedure, a pure crystalline enzyme-benzoate complex can be obtained from hog kidney. From this complex, holoenzyme and apoenzyme can be prepared. Purification can be carried out at room temperature unless otherwise stated. The holoenzyme is prepared from the crystalline benzoate complex by repeated expulsion of the benzoate from the complex with an excess of D-alanine. It is preferable to carry out this procedure in an ice bath. The purple complex, a reaction intermediate of D-amino acid oxidase, is crystallized from the mixture of the holoenzyme, the substrate, Dalanine, and the products, pyruvate and ammonia, under anaerobic conditions. The crystals consist of equimolar amounts of the enzyme (on the molecular basis of 50,000) and the substrate moiety which is readily converted into the products on aeration.