Superoxide Dismutases

Abstract

Superoxide dismutase was purified from pea (Pisum sativum L., cv. Wando) seeds and corn (Zea mays L., cv. Michigan 500) seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoreticaUy distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase. Superoxide dismutase accounted for 1.6 to 2.4% of the water-soluble protein in seedlings of corn, peas, and oats (Avena sativa L., cv. Au Sable). The superoxide dismutase activity per plant and per milligram water-soluble protein considerably increased during germination of oats and during greening and hook opening of peas. In a previous study, considerable amounts of SOD3 were found to be present in roots, shoots, seeds, and seed parts of oats, corn, and peas (4). Electrophoresis indicated multiple forms of the enzyme. Significant differences in quantity and forms of the enzyme were observed between species and between organs within a species. The objective of this study was to substantiate further the occurrence of the enzyme in higher plants and to examine the observed differences between species. For this purpose the enzyme was purified. Changes of SOD activity during seedling growth were also studied. MATERIALS AND METHODS Enzyme Purification. Unless otherwise stated, all operations were performed at 0 to 4 C. Dry pea seeds (Pisum sativum L., cv. Wando) were soaked in distilled H20 for about 15 hr. The resulting 1650 g wet weight was crushed with an electric mortar and pestle and homogenized with 1 liter of 0.1 M K2HPO4 in a Waring Blendor. The resulting pH was 7.5. After stirring, the slurry was filtered/squeezed through six layers of cheesecloth. The filtrate was centrifuged twice at 13000g for 30 min in a Sorvall RC2-B refrigerated centrifuge. The supernatant was subjected to the Tsuchihashi (chloroform-ethanol) treatment essentially as described by Weisiger and I Michigan Agricultural Experiment Station Journal Article No. 7765. 2 This paper represents part of the Ph.D. dissertation of C. N. G. Abbreviations: SOD: superoxide dismutase (EC 1 .15.1.1); NBT: pnitro blue tetrazolium chloride. Fridovich (10). None of the pea SOD enzymes is inactivated by this treatment. The supernatant was mixed with 0.25 volume of ethanol and 0.15 volume of chloroform and stirred for 15 min. It was then clarified by centrifugation at 13000g for 15 min. Chloroform that was separated out during centrifugation was removed by suction. The supernatant was decanted, solid K2HPO4 (20 g/l) was added, and the two phases were separated after 30 min. The less dense, ethanol-rich phase was collected, chilled, and centrifuged at -15 C. Additional chloroform separating out during centrifugation was removed by suction, and the ethanolic phase was decanted. Chilled acetone (0.5 volume) was added to the ethanolic phase while stirring. The precipitate was removed by centrifugation. Additional acetone (1.5 volume) was added to the supernatant, and the second precipitate was collected and redissolved in a minimal volume of 0.05 M K-phosphate (pH 7.8). Solid (NH4)2SO4 was added to the supernatant to bring it to 70% saturation. After 1 hr, the second precipitate was collected, and resuspended in and dialyzed against 0.1 M KCl, 5 mm K-phosphate, and 10 /LM EDTA (pH 7.8). The enzyme was further purified by gel exclusion chromatography. It was first applied on a Sephadex G-100 column (2 x 90 cm) equilibrated with the dialysis buffer. The void volume of the column was 74.5 ml and the flow rate 0.2 ml/min. This column was calibrated with proteins of known mol wt (9). Fractions with a specific activity greater than 300 units/mg protein were pooled and salted out from 70% saturated (NH,)2SO solution. The precipitate was collected by centrifugation, dissolved in a small volume of distilled H20, and dialyzed against the eluting buffer. It was then applied on a column (1 x 60 cm) of Bio-Gel P-30 equilibrated with the same buffer. Some impurities of higher mol wt were removed by this column. Fractions whose specific activity exceeded 700 units/mg were pooled and concentrated as above. The enzyme was rechromatographed on the same BioGel P-30 column. Impurities of slightly lower mol wt were partially separated from the enzyme. Only the two fractions of maximum specific activity (around 2000 units/mg) were pooled this time. Additional impurities were removed by fractionation with chilled acetone. The most active fraction was obtained between 1.5 and 2 volumes of acetone. The precipitate from the last fraction was redissolved in 0.05 M K-phosphate (pH 7.8). Enzyme from corn (Zea mays L., cv. Michigan 500) seedlings was partially purified. The seeds were treated with 0.3% (w/v) captan 80W for 5 min and germinated for 7 days on moist paper towels in the dark at room temperature. The seed remnants were removed, and the seedlings (182 g) were rinsed with distilled H20 and cut into 1-cm sections with a stainless steel razor blade. The tissue was homogenized with 400 ml of 0.1 M K-phosphate and 0.1 mm EDTA (pH 7.8) in a Waring Blendor. After 1 hr in the cold and occasional stirring, the homogenate was filtered/ squeezed through four layers of cheesecloth. The filtrate was centrifuged twice at 13000g for 20 min. This supernatant was