Partial Purification of Aconitase

In 1936, Martius and Knoop (1) discovered an enzyme which catalyzed the following reactions: + Hz0 + Hz0 Citric acid +Z, cis-aconitic acid *&XZZZ isocitric acid -H20 -Hz0This enzyme was later named aconitase by Breusch (2).At equilibrium, the three substances, citric, aconitic, and isocitric acids, exist in the approximate molecular ratio of 89 : 3 : 8 (3).Jacobsohn and his associates (4, 5) have provided indirect evidence that "aconitase" may be composed of two separate enzymes, each specifically catalyzing a single reaction, but this point has never been definitely established.Johnson has studied the kinetics of aconitase as well m its relative activity in different tissues (6).Krebs and Eggleston (3) have contributed further information on the properties of aconitase, including data on various stabilizers and inhibitors of the enzyme.Although Ochoa has concentrated the enzyme by ammonium sulfate precipitation (7), he has not attempted to fractionate or purify it.Contrary to previous reports, aconitase may be precipitated from solution by alcohol, and even acetone, without loss of activity, provided the temperature is kept sufficiently low.Alcohol was used as a fractionating agent in the purification of the enzyme described below. MethodsCitric acid was determined by the method of Pucher, Sherman, and Vickery (8) as modified by Speck et al. ( 9).d-Isocitric and L-malic acids were determined by the polarimetric method as described by Krebs and Eggleston (10).Although protein concentrations are reported in mg. per cc. in Table I, the concentrations of protein during the course of aconitase fractionation