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Enzymatic Assay of Superoxide Dismutase

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

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


3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC

3.2 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.

3.3 XOD – Xanthine Oxidase

3.4 SOD – Superoxide Dismutase.

3.5 O2¯ - Superoxide Radical


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

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

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

It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

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


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

Continuous Spectrophotometric Rate Determination


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, Sigma-Aldrich Product Number , 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 4.0mg/ml solution of Ethylenediaminetetraacetic acid disodium salt dihydrate, Sigma-Aldrich Stock Number , ED2SS in purified water.

7.3.3    1.1 mM Cytochrome C Solution (Cyt C)
            Prepare a 14.6mg/ml solution of Cytochrome C, Sigma-Aldrich Product Number , C7752 in purified water.

7.3.4    0.108 mM Xanthine Solution (Xanthine)    Dissolve 1.64mg of Xanthine, Sigma-Aldrich Product Number , X0626 in 90 milliliters of purified water.    With stirring, add small amounts of 1N KOH until all of the xanthine has dissolved    Quantitatively transfer the solution to a 100ml volumetric flask and qs to 100 milliliters with purified water.

7.3.5    Xanthine Oxidase Enzyme Solution (XOD)    Prepare a solution containing approximately 5units/ml of xanthine oxidase, Sigma-Aldrich Product Number , X1875 in cold purified water. Place on ice.    Immediately before use in Section 7.4.3., prepare a solution in cold purified water containing 0.05 units/mL of xanthine oxidase using Reagent This concentration may need to be adjusted to meet the requirements of Section 7.4.3.

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


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

Purified Water 23.0
Reagent 7.3.1 (Buffer) 25.0
Reagent 7.3.2 (EDTA) 1.0
Reagent 7.3.3 (CytoC) 1.0
Reagent 7.3.4 (Xanthine) 50.0

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

  Blank XOD
Reagent 7.4.2 (Cocktail) 2.80 2.80    Equilibrate to 25°C using a suitably thermostated spectrophotometer. Monitor the Absorbance at 550nm until constant, then add:

Purified Water 0.20 0.10
Reagent (XOD) ----- 0.10    Mix by inversion and record the increase in absorbance at 550nm 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 and repeat Section 7.4.3.

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

  Blank Uninhibited Test-1 Test-2 Test-3
Reagent 7.4.2 (Cocktail) 2.80 2.80 2.80 2.80 2.80
Purified water 0.20 0.10 ----- 0.01 0.02
Reagent 7.3.6 (SOD) ----- ----- 0.10 0.09 0.08

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

Reagent (XOD) ----- 0.10 0.10 0.10 0.10

7.4.6    Mix by inversion and record the increase in absorbance at 550nm 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 fall within 40-60% of the uninhibited rate. Any value outside this range is considered invalid.


7.5.1 Percent Inhibition: (ΔA550nm/min Uninhibited - ΔA550nm/min Inhibited)(100)
(ΔA550nm/min Uninhibited - ΔA550nm/min Blank)

  Units/ml Enzyme: (Percent Inhibition)(DF)

    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

In a 3.00 ml reaction mix, 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


8.1 McCord, J. M. and Fridovich, I. (1969) J. Biol. Chem. 244, 6049 6055

8.2 Procedure updated from SOP 10-30-6299 and OP SPCYTO01.


Review, approvals and signatures for this document will be generated electronically using the EDMS. Print a “For Use” copy if hardcopy with signature verification is required.

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