XOD (Xanthine oxidase)
This enzyme is useful for enzymatic determination of inorganic phosphorus, 5′-nucleotidase and adenosine deaminase.
Preparation and specification
|
Appearance |
Reddish brown amorphous powder,lyophilized |
|
Protein purity |
≥80% (from SDS-PAGE) |
|
Activity |
≥10 U/mg solid |
|
Adenosine deaminase |
≤0.001% |
|
Catalase |
≤5% |
|
Uricase |
≤0.001% |
|
Phosphatase |
≤0.001% |
|
Purine nucleoside phosphorylase |
≤0.005% |
Properties
|
EC number |
1.1.3.22 |
|
Molecular weight |
160 kDa (Gel) |
|
Isoelectric point |
4.0 |
|
Michaelis constants |
4.5× 10-5 M (Xanthine)， 7.6× 10-5 M (Hypoxanthine) |
|
Inhibitors |
Ag+, Hg2+ |
|
Optimum pH |
7.5-8.0 Fig. 1 |
|
Optimum temperature |
65 ℃ Fig. 2 |
|
pH stability |
pH 6.5-9.0 (25 ℃, 16 h) Fig. 3 |
|
Thermal stability |
Below 55 ℃ (pH 7.0, 30 min) Fig. 4 |
|
Storage stability |
At least one year at -25 ～- 15 ℃ Fig. 5 |
|
Stabilizers |
BSA, sodium glutamate |
Storage conditions
Store at -25～- 15℃, valid for 1 year
Figures
Assay principle
Unit definition
One unit (U) is defined as the amount of enzyme which generates 1 μmol of uric acid per minute at 37 ℃ under the conditions described below.
Reagents preparation
Reagent I: 100 mM, pH 7.5 Tris-HCl buffer.
Reagent II: 25 mM NaOH solution.
Reagent III: 10 mM xanthine (dissolved by Reagent II).
Reagent IV: 1 mM Oxonic acid potassium salt solution. Enzyme dilution buffer: 50 mM pH 7.5 Tris-HCl buffer.
Sample: The enzyme was diluted to 0.1-0.2 U/ml with enzyme dilution buffer.
Procedure
1. Add 2.24 ml Reagent I, 0.08ml Reagent III and 0.08ml Reagent IV to the 3 mL cuvette and equilibrate at 37 ℃ for 5 min.
2. Add 0.1 mL enzyme solution to the 1mL cuvette and mix.
3. Record the ΔAs at 293 nm in 1 minute in a spectrophotometer thermostated at 37 ℃. At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As – ∆Ab
Calculation
2.5: Total volume (ml)
0.1: enzyme volume (ml)
df: dilution factor
C: Enzyme concentration (mg/ml)
1.0: Light path length (cm)
12.5: Millimolar extinction coefficient of uric acid under 293nm (cm2 /μmol)
