The Oxidizing Enzymes of Sugarcane: Cytochrome C Oxidase

Abstract

A study was made of the distribution and properties of cytochrome C oxidase in sugarcane. The enzyme was extracted from fresh root tissues of variety P.R. 980 with water and precipitated with ammonium sulfate. Cytochrome C oxidase was assayed spectrophotormetrically at 550 mu by measuring the optical-density decline of a buffered solution of reduced ox-heart cytochrome C and enzyme. Fractionation of cane extracts with ammonium sulfate showed that cytochrome C oxidase is precipitated primarily between 80- and 95-percent saturation. This corresponds to the range of little or no tyrosinase precipitation, indicating that tyrosinase may act to mask the action of cytochrome C oxidase in crude cane preparations. Richest source of the oxidase was root tissue, although moderate activity was also present in the 80- to 95-percent fraction of meristem extracts. Some activity was likewise obtained with sheath and node preparations. Cytochrome C oxidase was stable for about 24 hours when maintained in dilute cysteine-HCl solution at 2°C. More than 50- percent of the activity was lost after 9 days. The enzyme was not adversely affected by freezing, it was totally inactivated by boiling for 5 minutes. Optimum temperature lay in the range of 36° to 40°C. Optimum pH was about 4.3. Cytochrome C oxidase activity was plotted as a first-order reaction with reference both to enzyme concentration and time. Iron appeared to be a limiting factor in the standard root preparations. Km was 4.5 X 10-5 mols of reduced cytochrome C per liter. Both mercury and cysteine-HCl stimulated the enzyme at the concentration 2.0 umols/ml. of digest. Boron and nitrate stimulated to a lesser extent at the same concentration, and cyanide caused total inhibition. Carbon monoxide did not inhibit when tested under normal light conditions. Dialysis against distilled water caused 70-percent activity decline within 2 hours, and almost total inactivation by 44 hours. Cysteine-HCl served to reactivate the enzyme. Iron, increased activity of the dialyzed preparation over that of the undialyzed control, indicating that this element is essential for maximum activity. Significance and potential roles of cytochrome C oxidase in sugarcane were discussed.