Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts / F. PAOLETTI; A. MOCALI; D. ALDINUCCI; A. CAPARRINI. - In: ANALYTICAL BIOCHEMISTRY. - ISSN 0003-2697. - STAMPA. - 154:(1986), pp. 536-541. [10.1016/0003-2697(86)90026-6]

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts

PAOLETTI, FRANCESCO;MOCALI, ALESSANDRA;
1986

Abstract

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.
1986
154
536
541
F. PAOLETTI; A. MOCALI; D. ALDINUCCI; A. CAPARRINI
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/309187
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