Improved preparation of bovine liver rhodanese.

Chemical studies of rhodanese (thiosulfate : cyanide sulfurtransferase, EC 2.8.1.1) in this laboratory have prompted the development of a modified procedure for preparing the crystalline enzyme. The new method, which is based on the earlier procedure of S6rbo (1) as modified by Westley et al. (2-4), permits isolation in apparently monodisperse form of nearly 50% of the total rhodanese activity of liver extracts. Furthermore, the changes introduced facilitate work on a larger scale so that routine preparation of more than 600 mg of crystalline enzyme at one time is practicable without special equipment. Enzymic activity was measured as previously reported (5). Protein concentration was determined by a modified biuret method (6) with crystalline rhodanese as standard. All chemicals used were of the standard reagent grade. With the exception of the 1 M acids, all solutions used in the following procedures conta,ined 1 mM Na&03 in addition to the stated constituents. All fractionation procedures were carried out at O-5”. Step I-Ten pounds of bovine liver (previously frozen and thawed) were homogenized in a l-gallon capacity Waring Blendor in three batches, each with 1,600 ml of 0.1 M glycine acetate buffer, pH 5.0. The homogenate was centrifuged for 30 min at 14,000 x g and the residue was extracted with 8 liters of the same buffer. Step %-To the combined extracts solid (NH&SO4 was added mechanically with stirring to 0.40 M and the pH was then adjusted to 3.8 with cold 1 M HCl. The ammonium sulfate concentration was raised to 1.4 M, and the solution was allowed to stand for at least 3 hours to permit complete precipitation. After removal of the precipitate by centrifugation, the ammo-