The copper of ascorbic acid oxidase; experiments with an ion exchange resin.

Recent investigations tend heavily to support the view that ascorbic acid oxidase is a specific enzyme containing copper as the prosthetic group of a blue-green conjugated protein having a molecular weight of about 150,000 (1, 2). Little is known at the present time concerning either the nature of the copper-protein bond in the enzyme or the function of the enzyme copper in the catalytic process. In contemplating the use of a radioactive isotope of copper as a tool for gaining more information about the copper-protein bond in ascorbic acid oxidase, it became apparent that one of the major requirements was the development of a rapid and quantitative method for separation of the enzyme from free ionic copper in aqueous solutions. Because of the relatively short half life of radioactive CuB4 (12.8 hours) (3), the simple dialysis process was too slow. Preliminary experiments with electrodialysis and protein precipitation techniques resulted in an unsatisfactory recovery of the enzyme activity. For these reasons, experimental work was undertaken to test the feasibility of using an ion exchange resin for quantitatively removing free ionic copper from ascorbic acid oxidase solutions. As revealed by the results described below, a resin which proved to be completely satisfactory for such separations, without injury to the enzyme, was Amberlite IR-100, analytical grade (4, 5). No attempt was made to evaluate all of the possible ion exchange resins.