胆固醇氧化酶是一种含有 FAD 的细菌黄素氧化酶，常用于生化研究。胆固醇氧化酶可以催化胆固醇（和其他甾醇）的 C(3)-OH 基团氧化生成 cholest-5-en-3-one，并将其异构化为 cholest-4-en-3-one。
技术指标
| 外观： | 黄色无定型冻干粉末 |
|---|
| 蛋白纯度： | ≥90% |
| 比活性： | ≥8 U/mg 酶粉 |
| 过氧化氢酶： | ≤0.001% |
| 胆固醇酯酶： | ≤0.01% |
| 葡萄糖氧化酶： | ≤0.01% |
| ATP 酶： | ≤0.005% |
酶学性质
| 来源： | 微生物 | |
| 酶学分类： | EC 1.1.3.6 | |
| 分子量： | 60 kDa (SDS-PAGE) | |
| 等电点： | 5.6 | |
| Km 值： | 3.3×10⁻⁴ M (Cholesterol) | |
| 抑制剂： | SDS、Ag⁺、Zn²⁺、Hg²⁺ | |
| 最适 pH： | 7.0 | 图 1 |
| 最适温度： | 50℃ | 图 2 |
| pH 稳定性： | 5.5-10（25℃，16 h） | 图 3 |
| 热稳定性： | 40℃以下稳定（pH 7.0，30 min） | 图 4 |
| 储存稳定性： | -25~-15℃静置保存 12 个月保持 90% 以上活性 | 图 5 |
| 保护剂： | BSA 以及非离子型表面活性剂 | |
用途
用于高、低密度脂蛋白胆固醇试剂的研发和大量配制。
活性测定方法
原理
反应生成的醌亚胺（Quinoneimine dye）的量可用分光光度计在 555 nm 检测。
酶活定义
单位酶活定义为在下述条件下，每分钟催化生 成 1 μmol H₂O₂ 所需的酶量。
试剂准备
试剂 I：0.1 M 磷酸钾缓冲液，pH 6.5。
试剂 II：在 500ml 烧杯中加入 5 mL Triton X-100 和 0.5 g 胆固醇，用保鲜膜密封，用微波炉加热至胆固醇完全溶解。缓慢加入 90 mL 双蒸水，加热并使其沸腾 30 - 60s，溶液将变浑浊。用自来水淋洗使溶液冷却，冷却后的溶液会变澄清，若冷却后未变澄清则需要重新加热使其完全溶解。在澄清的溶液中加入 0.4 g 胆酸钠，用双蒸水定容至 100 mL。定容后用 0.22um 滤膜过滤，此溶液在室温下一周内稳定。如果该溶液变浑浊，可轻微加热使其澄清。
试剂 III：纯水配制 100 U/mL POD。
试剂 IV：50 mM 4-AA 溶液（1.016 g 4-AA 溶于 100 mL UP 水）。
试剂 V：50 mM TOOS 溶液（1.477 g TOOS 溶于 100 mL UP 水）。
酶稀释液：20 mM 磷酸钾缓冲液，pH 7.0，含 0.2% BSA。
样品：用酶稀释液将酶稀释至 0.05 - 0.2 U/mL。
按如下配制反应混合物：
试剂 I：53 mL
试剂 II：7 mL
试剂 III：5 mL
试剂 IV：3 mL
试剂 V：3 mL
双蒸水：19 mL
共 90 mL，可用于 100 次反应
操作步骤
1. 将反应混合物置于 37℃水浴锅预热；
2. 向 1 mL 比色皿中加入 0.9 mL 反应混合物。
3. 向反应混合物中加入 0.1 mL 待测酶液，混匀。
4. 37℃反应，用分光光度计在 555 nm 检测样品 1 min 内的吸光度变化（ΔAs）
* 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（ΔAb）
ΔA = ΔAs - ΔAb
活力计算
Vt：反应液总体积（1 mL）；
Vs：酶液体积（0.1 mL）；
df：稀释倍数；
C：酶浓度（mg/mL）；
1.0：光路长度（cm）；
39.2：标准反应条件下，生色基团在 555nm 处毫摩尔吸光系数（cm²/μmol）。
Cholesterol oxidase (CHO) is a bacterial flavin oxidase containing FAD, commonly used in biochemical research. Cholesterol oxidase catalyzes the oxidation of the C(3)-OH group of cholesterol (and other sterols) to cholest-5-en-3-one and isomerizes it to cholest-4-en-3-one.
Technical Indicators
Appearance: Yellow amorphous lyophilized powder
Protein Purity: ≥90%
Specific Activity: ≥8 U/mg enzyme powder
Catalase: ≤0.001%
Cholesterol Esterase: ≤0.01%
Glucose Oxidase: ≤0.01%
ATPase: ≤0.005%
Enzymatic Properties
| Source: | Microorganism |
| Enzyme Classification: | EC 1.1.3.6 |
| Molecular Weight: | 60 kDa (SDS-PAGE) |
| Isoelectric Point: | 5.6 |
| Km Value: | 3.3×10⁻⁴ M (Cholesterol) |
| Inhibitors: | SDS, Ag⁺, Zn²⁺, Hg²⁺ |
| Optimal pH: | 7.0 (Fig.1) |
| Optimal Temperature: | 50℃ (Fig.2) |
| pH Stability: | 5.5-10 (25℃, 16 h) (Fig.3) |
| Thermal Stability: | Stable below 40℃ (pH 7.0, 30 min) (Fig.4) |
| Storage Stability: | Stable for 12 months at -25~-15℃ with over 90% activity (Fig.5) |
| Protectant: | BSA and non-ionic surfactants |
Applications
It is used in the research and development and large-scale formulation of high and low-density lipoprotein cholesterol reagents.
Assay Method
Principle
The amount of quinoneimine dye generated by the reaction can be detected by a spectrophotometer at 555 nm.
Unit Definition
One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol H₂O₂ per minute under the following conditions.
Reagent Preparation
Reagent I: 0.1 M potassium phosphate buffer, pH 6.5.
Reagent II: In a 500 ml beaker, add 5 mL Triton X-100 and 0.5 g cholesterol, seal with plastic wrap, and heat in a microwave until the cholesterol is completely dissolved. Slowly add 90 mL double-distilled water, heat and bring to a boil for 30 - 60 s, the solution will become turbid. Rinse with tap water to cool the solution, the cooled solution will become clear. If it does not become clear after cooling, it needs to be reheated until completely dissolved. Add 0.4 g sodium taurocholate to the clear solution, dilute to 100 mL with double-distilled water. After constant volume, filter through a 0.22 μm filter membrane. This solution is stable at room temperature for one week. If the solution becomes turbid, it can be slightly heated to clarify.
Reagent III: 100 U/mL POD prepared with pure water.
Reagent IV: 50 mM 4-AA solution (1.016 g 4-AA dissolved in 100 mL UP water).
Reagent V: 50 mM TOOS solution (1.477 g TOOS dissolved in 100 mL UP water).
Enzyme Dilution Buffer: 20 mM potassium phosphate buffer, pH 7.0, containing 0.2% BSA.
Sample: Dilute the enzyme with the enzyme dilution buffer to 0.05 - 0.2 U/mL.
Prepare the reaction mixture as follows:
Reagent I: 53 mL
Reagent II: 7 mL
Reagent III: 5 mL
Reagent IV: 3 mL
Reagent V: 3 mL
Double-distilled water: 19 mL
Total volume: 90 mL, which can be used for 100 reactions
Operating Procedures
1. Preheat the reaction mixture in a 37℃ water bath;
2. Add 0.9 mL of the reaction mixture to a 1 mL cuvette;
3. Add 0.1 mL of the sample to be tested into the reaction mixture and mix well.
4. React at 37℃, and detect the absorbance change (ΔAₛ) of the sample within 1 min at 555 nm with a spectrophotometer.
* Replace the enzyme with enzyme dilution buffer, and follow the same steps. The absorbance of the resulting solution is the blank absorbance (ΔAᵦ).
ΔA = ΔAₛ - ΔAᵦ
Calculation
Vt: Total volume of the reaction solution (1 mL);
Vs: Volume of the enzyme solution (0.1 mL);
df: Dilution factor;
C: Enzyme concentration (mg/mL);
1.0: Path length (cm);
39.2: Molar extinction coefficient of the chromophore at 555 nm under standard reaction conditions (cm²/μmol).
ZJ25F1129800
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