Enzymatic Assay of Superoxide Dismutase

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) ΔA_550nm 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, A_550nm, 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:

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:

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

	Blank	XOD
Reagent 7.4.2 (Cocktail)	2.80	2.80

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

Purified Water	0.20	0.10
Reagent 7.3.5.2 (XOD)	-----	0.10

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:

	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 550 nm until constant, and then add:

Reagent 7.3.5.2 (XOD)

-----

0.10

0.10

0.10

0.10

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 ΔA_550nm for each inhibited test should be within 40-60% of the uninhibited rate. Any value outside this range is considered invalid.

7.5 CALCULATIONS

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

	Units/mL Enzyme:	(Percent Inhibition)(DF)
		(50%)(0.10)

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