用于尿酸试剂的研发和大量配制。
酶学性质
来源：Arthrobacter globiformis
分类：EC 1.7.3.3
分子量：40 kDa (SDS-PAGE)
等电点：5.1
Km 值：2.1x10⁻⁵ M (Urate)
抑制剂：Fe³⁺, Co²⁺, Cu²⁺, Mn²⁺, Zn²⁺
最适 pH：8.5 图 1
最适温度：37℃ 图 2
pH 稳定性：5.5-10.0 (25℃, 16 h) 图 3
热稳定性：60℃以下稳定 (pH 8.5,30 min) 图 4
稳定性：-25~-15℃静置保存 12 个月保持80%以上活性 图 5
保护剂：硼酸盐、EDTA及非离子型表面活性剂
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
活性测定方法
1、原理
尿酸（Uric acid）的量可用分光光度计在290 nm下检测。
2、酶活定义
单位酶活定义为在下述反应条件下，每分钟氧化 1μmol 尿酸所需的酶量。
3、试剂准备
酶稀释液：在800 mL双蒸水中溶解3.09 g硼酸，0.37 g EDTA·Na₂·2H₂O 和 0.01 g Triton X-100，用 NaOH调节pH至8.5，用双蒸水定容至 1000 mL。
试剂 I：0.01%尿酸母液（称取0.01 g尿酸， 用酶稀释液定容至 100 mL）。
试剂 II：0.001%尿酸溶液（取10 mL试剂 I， 用酶稀释液定容至 100 mL）。
样品：用酶稀释液将酶稀释至0.25-0.50 U/mL。
4、操作步骤
4.1 将试剂 II 置于 37℃水浴锅预热。
4.2 向 3 mL 比色皿中加入 2 mL 试剂 II，0.5 mL双蒸水。加入0.5 mL待测酶液，混匀。
4.3 37℃反应，用分光光度计在290 nm检测 样品1 min 内的吸光度变化（∆As）。
*用酶稀释液替代酶液，其它步骤相同，所得溶液吸光度为空白吸光度（∆Ab）
∆A=∆As-∆Ab
5、 活力计算
3.0：反应液总体积（mL）；
0.5：酶液体积（mL）；
1.0：光路长度 (cm)；
df：稀释倍数；
C：酶浓度( mg/mL)；
5.0：活力换算系数；
12.2：尿酸在290 nm处毫摩尔吸光系数(cm2/ μmol)。
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).
ZJ26F0939154
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