BOVINE ADRENAL GLUCOSE‐6‐PHOSPHATE DEHYDROGENASE

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Abstract
We have developed a new procedure for the purification of glucose‐6‐phosphate dehydrogenase from bovine adrenals. Three kg of whole adrenals are homogenized and fractionated by precipitation with ammonium sulfate, adsorption and elution from calcium phosphate gel, chromatography on cellulose phosphate, chromatography on DEAE‐Sephadex, gel filtration on Sephadex C‐J50, and crystallization from ammonium sulfate solution. The procedure requires about three weeks and the yield is 24 mg of enzyme. Approximately half of the original enzyme activity is lost by inactivation in spite of considerable effort to minimize these losses. Analysis of the final product by sedimentation velocity displays a single Gaussian peak, and the boundary spreading can be attributed almost entirely to diffusion, thus a high degree of mass homogeneity is demonstrated. The sedimentation coefficient converted to standard conditions, Szo.w is 9.47S. This is consistent with a molecular weight of 190,000. The enzyme also displays a single band when submitted to electrophoresis on cellulose acetate strips. The activity of 6‐phosphogluconate dehydrogenase disappears early in the fractionation procedure.