Application: For the R&D and large-scale preparation of uric acid reagents.
Enzymatic Properties
| Source: | Arthrobacter globiformis | |
| Classification: | EC 1.7.3.3
| |
| Molecular Weight: | 40 kDa (SDS-PAGE) | |
| Isoelectric Point (pI): | 5.1
| |
| Km Value: | 2.1 × 10⁻⁵ M (Urate)
| |
| Inhibitors: | Fe³⁺, Co²⁺, Cu²⁺, Mn²⁺, Zn²⁺
| |
| Optimal pH: | 8.5
| Fig.1 |
| Optimal Temperature: | 37℃ | Fig.2 |
| pH Stability: | 5.5-10.0 (25℃, 16 h)
| Fig.3 |
| Thermal Stability: | Stable below 60℃ (pH 8.5, 30 min)
| Fig.4 |
| Storage Stability: | ≥80% activity retained after 12 months of static storage at -25~-15℃
| Fig.5 |
| Protectants: | Borate, EDTA, and non-ionic surfactants |
Buffer solution: pH 4.5-5.5, NaAc-HAc; pH 6.0-8.0,
Na-phosphate; pH 8.0-9.0, Tris-HCl; pH 9.0-10.0,
Glycine-NaOH.
Enzyme concentration: 1 mg/mL
Reaction in 50 mM Boric acid buffer pH 8.5.
Enzyme concentration: 1mg/mL
25°C, 16 h-treatment with 50 mM buffer solution: pH
4.5-5.5, NaAc-HAc; pH 6.0-8.0, Na-phosphate; pH
8.0-9.0, Tris-HCl; pH 9.0-10, Glycine-NaOH. Enzymeconcentration: 1mg/mL
30min-treatment with 50 mM Boric acid buffer,pH 8.5.
Enzyme concentration: 1mg/mL
Activity Assay Method
1. Principle
The concentration of uric acid can be determined by spectrophotometry at 290 nm.
2. Definition of Enzyme Activity
One unit (U) of enzyme activity is defined as the amount of enzyme required to oxidize 1 μmol of uric acid per minute under the specified reaction conditions.
3. Reagent Preparation
Enzyme Dilution Buffer: Dissolve 3.09 g of boric acid, 0.37 g of EDTA·Na₂·2H₂O, and 0.01 g of Triton X-100 in 800 mL of double-distilled water. Adjust the pH to 8.5 with NaOH, then bring the final volume to 1000 mL with double-distilled water.
Reagent I: 0.01% uric acid stock solution (weigh 0.01 g of uric acid, dissolve and dilute to 100 mL with Enzyme Dilution Buffer).
Reagent II: 0.001% uric acid working solution (take 10 mL of Reagent I, dilute to 100 mL with Enzyme Dilution Buffer).
Test Sample: Dilute the enzyme solution to 0.25-0.50 U/mL with Enzyme Dilution Buffer.
4. Procedure
4.1. Preincubate Reagent II in a 37℃ water bath.
4.2. Add 2 mL of Reagent II and 0.5 mL of double-distilled water to a 3 mL cuvette. Then add 0.5 mL of the test sample and mix thoroughly.
4.3. Incubate the reaction mixture at 37℃, and measure the absorbance change (∆As) of the sample at 290 nm within 1 minute using a spectrophotometer.
Blank Control: Replace the enzyme solution with Enzyme Dilution Buffer and follow the same steps to obtain the blank absorbance change (∆Ab).
∆A = ∆As - ∆Ab
5. Activity Calculation
3.0: Total volume of the reaction mixture (mL).
0.5: Volume of the enzyme solution added (mL).
1.0: Light path length (cm).
df: Dilution factor of the enzyme solution.
C: Concentration of the enzyme solution (mg/mL).
5.0: Activity conversion factor.
12.2: Molar extinction coefficient of uric acid at 290 nm (cm²/μmol).
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