Reaction of coppersuperoxide dismutase with hydrogen peroxide: a possible source of Heterogeneity?

Abstract

The reaction of hydrogen peroxide with copperzinc bovine superoxide dismutase at ratios of 0.2 to 4.0 per active site at pH 10.0 results in the formation of distinct electrophoretic forms of the enzyme. The mobilities of these forms are identical to the mobilities of the forms present in heterogeneous, untreated native samples in two different electrophoretic systems, pH 6.96 (TRIS-acetic acid) and pH 8.38 (TRIS-glycine) (Fig.1.). Increasing ratios of hydrogen peroxide result in an apparent increase in this heterogeneity in addition to progressive inactivation (Fig. 2). Inactivation of the dismutase and loss of histidine was reported by Hodgson and Fridovich [1]. Furthermore this reaction results in the loss of copper from the dismutase at all ratios of added peroxide and the loss of zinc at the higher ratios (Table I). The loss of copper parallels the increase in the faster moving species suggesting the possibility that this species is copperdeficient. However, in the pH 6.96 electrophoretic system, the mobility of this faster form does not coincide with the mobility of a copper-deficient form prepared by reconstituting the apoenzyme with equimolar copper and zinc [2]. If it is copper-deficient, the faster electrophoretic form must be modified by the reaction with peroxide. It should be noted that this form is not capable of giving rise to native enzyme upon the addiction of copper and zinc. In addition the reaction with peroxide forms distinct electrophoretic forms of the enzyme at the more physiological pH 7.0. Analysis of several purified preparations of superoxide dismutase have indicated that a ▪ ▪ lower copper content is correlated with an increased heterogeneity (Table II). For all samples listed but one, this heterogeneity was not altered by the addition of copper and zinc. In view of these observations we would like to suggest that the reaction with hydrogen peroxide in vivo may be the source of the heterogeneity so often seen in purified preparations of the copperzinc enzyme. Hydrogen peroxide is the product of the enzyme-catalyzed dismutation reaction of superoxide anion and its reaction with the dismutase, though slow, could lead to the accumulation of altered proteins t001 Loss of Copper and Zinc from Superoxide Dismutase in the Reaction with Hydrogen Peroxide at pH 10.0 H 2O 2 Copper Zinc site Enzyme Enzyme 0.0 1.30 1.92 0.49 1.23 1.94 0.98 1.18 1.69 1.46 1.16 1.79 1.96 1.00 1.23 2.96 0.87 3.96 0.85 1.08 t002 Copper Content and Heterogeneity in Preparations of Bovine Superoxide Dismutase Sample Copper %I %II %III Enzyme Sigma [1] 1.89 83 15 2 DDI 1.83 83 16 1 Sigma [2] 1.75 66 29 5 DDI 1.61 71 24 5 Malta 1.42 40 58 2 Sigma [2] dialyzed 1.30 52 34 14 that copurify with the native enzyme in classical isolation procedures.