1. Objective

To standardize a procedure for the enzymatic determination of superoxide dismutase.

2. Scope

This procedure applies to all products that have a specification for superoxide dismutase activity by enzymatic determination.

3. Definitions

  • Purified Water: water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC
  • Unit Definition: One unit will inhibit the rate of reduction of cytochrome c by 50% in a coupled system, using xanthine and xanthine oxidase at pH 7.8 at 25 °C in a 3.0 mL reaction volume. The xanthine oxidase concentration should produce an initial (uninhibited) ΔA550nm of 0.025 +/- 0.005 per minute.
  • XOD – Xanthine Oxidase
  • SOD – Superoxide Dismutase.
  • O2¯∙ - Superoxide Radical

4. Discussion

The superoxide radical is produced enzymatically by the reaction catalyzed by Xanthine Oxidase:
Xanthine + O2 + H2O XOD > Uric acid + O2¯∙ + H+

Oxidised cytochrome c is reduced by the superoxide radical. The rate of reduction is followed spectrophotometrically at 550 nm:
Cytochrome3+ c + O2¯ > Cytochrome2+ c + O2

Superoxide dismutase inhibits the reduction of cytochrome c by competing for the superoxide radical:
2 O2¯∙ + 2 H+ SOD > O2 + H2O2

5. Responsibilities

Analytical services laboratory personnel should follow this procedure as written.

6. Safety

Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS: T = 25 °C, pH = 7.8, A550nm, Light path = 1 cm

7.2 METHOD: Continuous spectrophotometric rate determination

7.3 REAGENTS:

7.3.1 216 mM Potassium Phosphate Buffer, pH 7.8 at 25 °C (Buffer): Prepare a 49.3mg/mL solution of potassium phosphate dibasic trihydrate (Product No. P5504) in purified water. Adjust the pH to 7.8 at 25 °C with 1 M KOH or 1 M HCl.

7.3.2 10.7 mM Ethylenediaminetetraacetic Acid Solution (EDTA): Prepare 4.0 mg/mL solution of Ethylenediaminetetraacetic acid disodium salt dihydrate (Product No. ED2SS) in purified water.

7.3.3 1.1 mM Cytochrome C Solution (Cyt C): Prepare a 14.6 mg/mL solution of Cytochrome C (Product No. C7752) in purified water.

7.3.4 0.108 mM Xanthine Solution (Xanthine): Dissolve 1.64 mg of Xanthine (Product No. X0626) in 90 mL of purified water. With stirring, add small amounts of 1 N KOH until all of the xanthine has dissolved. Quantitatively transfer the solution to a 100 mL volumetric flask and qs to 100 mL with purified water.

7.3.5 Xanthine Oxidase Enzyme Solution (XOD): Prepare a solution containing approximately 5 units/mL of xanthine oxidase (Product No. X1875) in cold purified water. Place on ice.

7.3.6 Immediately before use in the assay, prepare a solution in cold purified water containing 0.05 units/mL of xanthine oxidase using Reagent XOD. This concentration may need to be adjusted to meet the requirements of Section 7.4.3.

7.3.7 Superoxide Dismutase Enzyme Solution: Immediately before use, prepare a solution containing 10 units/mL of superoxide dismutase in cold purified water.

7.4 ASSAY PROCEDURE

7.4.1 Prepare a reaction cocktail by pipetting the following reagents (in milliliters) into a suitable container:

7.4.2 Mix and adjust the pH to 7.8 at 25 °C with 1 M HCl or 1 M KOH if necessary.

7.4.3 Xanthine Oxidase Check:

7.4.3.1 Pipette the following (in milliliters) into suitable cuvettes:

7.4.3.2 Equilibrate to 25 °C using a suitably thermostated spectrophotometer. Monitor the absorbance at 550 nm until constant, and then add:

7.4.3.3 Mix by inversion and record the increase in absorbance at 550 nm for approximately 5 minutes. The change in absorbance for the uninhibited versus the blank should be 0.025+/-0.005 for this reaction. If it is not, adjust the concentration of Reagent 7.3.6 and repeat Section 7.4.3.

7.4.4 Pipette (in milliliters) the following reagents into suitable cuvettes:

7.4.5 Equilibrate to 25 °C using a suitably thermostated spectrophotometer. Monitor the absorbance at 550 nm until constant, and then add:

7.4.6 Mix by inversion and record the increase in absorbance at 550 nm for approximately 5 minutes. Obtain the fastest linear rate over a one minute interval for the uninhibited reaction. Using this time interval, obtain the rates for each Test and Blank.

7.4.7 The ΔA550nm for each inhibited test should be within 40-60% of the uninhibited rate. Any value outside this range is considered invalid.

7.5 CALCULATIONS

DF = Dilution Factor
50% = Inhibition of the rate of cytochrome c reduction per the unit definition
0.10 = Volume (in milliliters) of enzyme used in each test

7.6 FINAL ASSAY CONCENTRATION :
In a 3 mL reaction, the final concentrations are 50 mM potassium phosphate, 0.1 mM ethylenediaminetetraacetic acid, 0.01 mM cytochrome c, 0.05 mM xanthine, 0.005 unit xanthine oxidase and 1 unit superoxide dismutase

Materials
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