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Dr. Bratko Filipič

Email: [email protected]

This assay for superoxide dismutase (SOD, EC 1.15.1.1) activity involves inhibition of nitroblue tetrazolium reduction, with xanthine-xanthine oxidase used as a superoxide generator. By using a reaction terminator, we can determine 40 samples within 55 mm. One unit of activity of pure bovine liver Cu,ZnSOD and chicken liver MnSOD was expressed by 30 ng and 500 ng of protein, respectively. The mean concentrations of Cu,ZnSOD as measured by this method in blood from normal adults were 242 (SEM 4) mg/L in erythrocytes, 548 (SEM 20) .tg/L in serum, and 173 (SEM 11) tgIL in plasma. The Cu,ZnSOD concentrations in serum and plasma of patients with cancer of the large intestine tended to be less and greater than these values, respectively, but not statistically significantly so.

Reagents

Standard SOD solution.

The stock solution consisted of 4 mg of Cu,ZnSOD from porcine erythrocytes (Shouchou Biochemical Reagent Co., Shouchou, China) dissolved in 50 mL of doubly distilled water, or 8 mg of bovine liver Cu,ZnSOD (Diagnostic Data, Mountain View, CA) dissolved in 8 mL of isotonic saline. This was refrigerated until use. Before use in the assay, the stored solution was diluted to 600 pgfL with doubly distilled water. To prepare standard MnSOD, we used chicken-liver MnSOD, purified by the method of Weisiger and Fridovich (14); its protein concentration was 1.1 gfL. We diluted this standard 10-fold just before use. Xanthine oxidase solution (15). We diluted 20 pL of xanthine oxidase (1 kU/g concentration as supplied, 20 U per 1.2 mL; Boehringer Mannheim GmbH, Mannheim, F.R.G.) to 2.0 mL with ice-cold 2 moLfL ammonium sulfate, freshly prepared. The final concentration of xanthine oxidase was then 167 U/L. SOD assay reagent. For a 40-tube assay, combine the following reagents in a 200-mL beaker and mix well: 40 mL of 0.3 mmol/L xanthine solution (Sigma Chemical Co., St. Louis, MO), 20 mL of 0.6 mmoIJL EDTA solution, 20 mL of 150 pniol/L mtroblue tetrazolium solution (NBT; Dongfong Biochemical Reagent Factory, Shanghai, China, or Sigma Chemical Co., St. Louis, MO), 12 mL of 400 mmol/L Na2CO3 solution, and 6 mL of bovine serum albumin (1 g/L; Serva Biochimica, F.R.G.). Preparation of samples (16).

We heparunized whole blood obtained by venipuncture, centrifuged (3000 rpm, 10 miii, 0-4 #{176}C), and carefully separated the plasma. We lysed 0.1 mL of erythrocytes with 0.9 mL of ice-cold water (4#{176}C), then removed hemoglobin (and MnSOD in plasma and serum) by adding 0.3 mL of chloroform and 0.5 mL of ethanol and vigorously vortex-mixing for 1 mm. We centrifuged the mixture at 18 000 x g for 60 miii. The supernatant fluid is diluted by a factor of 100, and 0.5 mL of the diluted solution is used to assay Cu,ZnSOD activities as described below. We treated the samples of plasma and serum with chloroform and ethanol also. We used for SOD assay 0.5 mL of the supernate after centrifugation at 18 000 x g for 60 mm.

Assay

Add 2.45 mL of the SOD assay reagent to each of 40 tubes, then add 0.5 mL of pure Cu,ZnSOD (0-270 ng), MnSOD (0- 5130 ng), or blood fraction to each tube. When MnSOD is to be measured, add NaCN (final concentration, 5 mmol/L) to the assay tube and pre-incubate for 30 mm to inhibit Cu,ZnSOD. The final volume of the reaction system is 3.0 mL and it contains, per liter, 0.1 mmol of xanthune, 0.1 mmol of EDTA, 50 mg of bovine serum albumin, 25 pmol of NBT, 9.9 nmol of xanthine oxidase, and 40 mmol of Na2CO3 (pH 10.2). Place a rack of 40 tubes into a water bath adjusted to 25#{176}C. Add 50 uL of xanthine oxidase solution to each tube at 30-s intervals. Incubate each tube for 20 miii (the time needed to add xanthine oxidase to the 40 tubes), then terminate the reaction by adding 1 mL of 0.8 mmol/L CuC12 solution per tube every 30 s (the interval between adding xanthine oxidase to the last tube and adding CuC12 to the first tube was 308). In this way, a 40-tube assay can be done within 40 miii, plus 15 mm for spectrophotometrically reading the absorbance of each sample. The production of formazan is determined at 560 nm. Under these conditions, the absorbance at 560 mu of the blank tube is about 0.25.

The percent inhibition is calculated as below: % inhibition = (Ablank - Asampie) /Asample

Draw the standard inhibition curve with the x-axis being the protein concentration and the y-axis the values of percent inhibition. One unit of SOD is defined as the amount ofprotein that inhibits the rate of NET reduction by 50%. Calculate the Cu,ZnSOD activity by comparison with the standard curve. To avoid determinate error, we constructed a standard curve for each assay run, using porcine erythrocyte SOD as standard.

Superoxide dismutase activity can be determined using colorimetric test (Biovision). Blood samples were collected using EDTA centrifuged at 1,000 × g for 10 min at 4ºC. The plasma layer was transferred to a new tube without disturbing the buffy layer and stored at -80ºC until ready for analysis. The buffy layer was removed from the red cell pellet. The erythrocytes were resuspended in 5X volume of ice-cold distilled water and centrifuged at 10,000 × g for 10 min to pellet the erythrocyte membranes. The supernatant was stored at -80ºC until ready for analysis. SOD activity was calculated in terms of units (U/ml).

Please check the attached PDF. Pyrogallol procedure is the best.