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

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

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) ΔA_550nm of 0.025 +/- 0.005 per minute.

3.3 XOD – Xanthine Oxidase

3.4 SOD – Superoxide Dismutase.

3.5 O2^¯∙ - Superoxide Radical

4. Discussion

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

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to 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)

7.3.1.1 Prepare a 49.3mg/mL solution of Potassium phosphate dibasic trihydrate, Product Number , P5504 in purified water.

7.3.1.2 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, 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, Product Number , C7752 in purified water.

7.3.4 0.108 mM Xanthine Solution (Xanthine)

7.3.4.1 Dissolve 1.64mg of Xanthine, Product Number , X0626 in 90 milliliters of purified water.

7.3.4.2 With stirring, add small amounts of 1N KOH until all of the xanthine has dissolved

7.3.4.3 Quantitatively transfer the solution to a 100 mL volumetric flask and qs to 100 milliliters with purified water.

7.3.5 Xanthine Oxidase Enzyme Solution (XOD)

7.3.5.1 Prepare a solution containing approximately 5units/mL of xanthine oxidase, Product Number , X1875 in cold purified water. Place on ice.

7.3.5.2 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 7.3.5.1. 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 ASSAY PROCEDURE

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:

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 550nm until constant, 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 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 7.3.5.2 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 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 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 ΔA_550nm for each inhibited test should fall 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 3.00 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. Approval

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.