NIPRO ENZYMESSUPEROXIDE DISMUTASE (SOD)[EC 1.15.1.1]
from Bacillus stearothermophilus
O2- + O2- + 2H+ ↔ O2 + H2O2
SPECIFICATIONStateSpecific activity
Contaminants
PROPERTIES
Molecular weight
Subunit molecular weight
Metal content
Optimum pH
pH stability
Isoelectric point
Thermal stability: Lyophilized: more than 9,000 U/mg protein: (as SOD activity = 100 %)Catalase: ca. 50,000: ca. 25,000: 1.5 g atoms of Mn per mole of enzyme: 9.5: 6.0 - 9.0: 4.5: No detectable decrease in activity up to 60 °C.

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for medicine, cosmetic material and nutrition or antioxidant.

< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)

NIPRO ENZYMESASSAYPrinciple
To determine the enzyme activity of cytochrome c reduction is measured by the following reactions.
Xanthine + O2
O2-Xanthine oxidase

Urate + O2- + H2O2

cytochrome cSOD

cytochrome c (red.)O2 + H2O2

Unit Definition
One unit of activity is defined as the amount of SOD required to inhibit the rate of reduction ofcytochrome C by 50 % at 30 °C.
Solutions
Ⅰ Buffer solution ; 75 mM Potassium phosphate buffer, pH 7.8
Ⅱ Xanthine solution ; 0.75 mM (0.010 g xanthine/50 mL N/250 NaOH)
Ⅲ Cytochrome c solution ; 0.15 mM (0.019 g cytochrome c/10 mL distilled water, Sigma-Aldrich Co.,
No. C-2506, from horse heart)Ⅳ EDTA solution ; 1.5 mM (0.028 g EDTA disodium salt∙2H2O/50 mL distilled water)
Ⅴ Xanthine oxidase (XOD) ; (from buttermilk, Sigma-Aldrich Co., No. X-1875) suspension in 2.3 M(NH4)2SO4 solution is diluted to 0.04 U/mL with distilled water. (prepare freshly)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to approx. 600 U/mL with 50 mM
potassium phosphate buffer, pH 7.5.
Procedure
1. Prepare the following reaction mixture and pipette 2.80 mL of reaction mixture and 0.005 mL ofenzyme solution into a cuvette.
SolutionⅠ 22.00 mL
SolutionⅢ 2.00 mL
SolutionⅡ 2.00 mL
SolutionⅣ 2.00 mL
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.20 mL of SolutionⅤ into the cuvette and mix.
4. Read absorbance change at 550 nm per minute for the linear portion of curve (ΔAbs•test)*.
5. Add 0.005 mL of SolutionⅠ in place of enzyme solution and measure the same above 4(ΔAbs•blank).
*Dilute enzyme solution with 50 mM potassium phosphate buffer, pH 7.5, because the decrease in theinitial rate should not fall outside the range of 40 to 60 % for the results to be valid.
Calculation
Volume activity (U/mL) =(ΔAbs•blank)(ΔAbs•test)

Specific activity (U/mg protein) =-1X601
1Volume activity (U/mL)
protein concentration (mg/mL)*

d.f. ; dilution factor*Protein concentration ; determined by Bradford's method
REFERENCE

X d.f.

NIPRO ENZYMES
1. Bridgen, J., Harris, J.I., and Kolb, E.; J. Mol. Biol., 105, 333 (1976)
2. Brock, C.J., Harris, J.I., and Sato, S.; ibid., 107, 175 (1976)
3. Brock, C.J., and Walker, J.E.; Biochemistry, 19, 2873 (1980)
4. Auffret, A.D., Blake, T,J., and Williams, D.H.; Eur. J. Biochem., 113, 333 (1981)
5. Atkinson, T., Banks, G.T., Bruton, C.J., Comer, M.J., Jakes, R., Kamalagharan, T., Whitak, A.R.,
and Winter, G.P.; J. Appl. Biochem., 1, 247 (1979)

Remaining activity (%)

Relative activity (%)

NIPRO ENZYMES

10050010050046578910 11 128910 11 12Fig. 2 pH stability
treated for 24 hr at 4 ℃ in the
Britton-Robinson buffer

Remaining activity (%)

Remaining activity (%)

7Fig. 1 pH profile

50306pH

100205pH△ acetate, ○ phosphate,● Tris-HCl, ▲ carbonate

044050607080

Temperature (℃)
Fig. 3 Thermal stability
treated for 15 min in 0.1 M potassium
phosphate buffer, pH 7.5

1005000153045Time (min)Fig. 4 Thermal stability
treated in 0.1 M potassium
phosphate buffer, pH 7.5○ 60 ℃, □ 70 ℃, ● 80 ℃60