乳酸脱氢酶是选用特定细菌菌株，经独特发酵工艺制备得到的乳酸脱氢酶制剂。
催化反应
D‑乳酸 + NAD⁺ + H₂O ⇌ 丙酮酸 + NADH + H⁺
规格与制剂信息
活性：乳酸脱氢酶活性 ≥ 100U/mg
杂酶：
NADH 氧化酶活性 ≤1×10⁻⁴ %
α‑酮戊二酸脱氢酶（α-KGDH）活性 ≤3×10⁻⁴ %
谷草转氨酶（GOT）活性 ≤5×10⁻⁴ %
谷丙转氨酶（GPT）活性 ≤5×10⁻⁴ %
外观：白色冻干粉末
添加剂：葡聚糖
产品特性
1. 分子量：64000（凝胶过滤法测定）
2. 等电点：4.3
3. Kₘ：6.7×10⁻⁴ M（丙酮酸）；7.6×10⁻⁴ M（NADH）
4. 最适 pH：5.0
5. pH 稳定性：5.5–8.5（30℃，保温 1 小时）
6. 最适温度：40℃
7. 热稳定性：≤35℃（pH7.0，保温 1 小时）
8. 应用：在临床诊断中，与配套酶偶联，用于多种代谢物（如 ATP、ADP、葡萄糖、肌酐、丙酮酸、乳酸、甘油）以及多种酶活性（如 GPT、PK、CPK）的酶法测定。
安全操作说明
1. 请勿吸入本品。
2. 若不慎接触皮肤或眼睛，立即用大量清水冲洗。
乳酸脱氢酶活性检测方法
原理
采用分光光度法在 340 nm 处检测 NADH 的消耗速率。
酶活单位定义
1 个酶活单位（U）定义：在下述测定条件下，每分钟氧化 1 μmol NADH 所需的酶量。
试剂
A. 0.1 mol/L Tris‑HCl 缓冲液（pH 7.8）
B. 0.01 mol/L 磷酸盐缓冲液（KH₂PO₄‑NaOH，pH 7.0）
C. 底物溶液
称取 12.4 mg β‑NADH，加入 20.5 mg 丙酮酸钠，用 A 缓冲液溶解，最后用 A 缓冲液定容至 100 mL。（避光冷藏可保存 5 天）
D. 酶液
称取本产品 L139685，用预冷的 B 磷酸盐缓冲液溶解；酶液稀释后，应保证 ΔOD / 分钟落在 0.015±0.005/min 区间。
操作步骤
向 10 mm 光径石英比色皿中加入 2.9 mL 底物溶液 C，25±0.5 ℃保温 5 min；再加入 0.1 mL 酶液 D，迅速混匀，体系维持 25±0.5 ℃。
加入酶液后，分别在第 2 min、第 5 min 读取 340 nm 吸光度（A2、A5）。
空白对照：用 B 缓冲液替代酶液 D，其余操作完全相同，得到空白吸光度 Ab2、Ab5。
计算公式
乳酸脱氢酶活性（U/mg）= [(A2−A5)−(Ab2−Ab5)] / 3 × 1 / 6.22 × 3.0 × n / 0.1
- 3：反应时长（min）
- 6.22：NADH 在 340 nm 处的毫摩尔消光系数
- 3.0：反应体系总体积（mL）
- 0.1：加入酶液体积（mL）
- n：酶液稀释倍数
Lactate Dehydrogenase is a lactate dehydrogenase preparation manufactured by a unique fermentation process using a selected strain of bacteria.
Catalysis
D‑Lactate + NAD⁺ + H₂O ⇌ Pyruvate + NADH + H⁺
Specification and Preparation
Activity: Lactate dehydrogenase activity ≥ 100 u/mg
Contaminants:
NADH oxidase activity ≤1×10⁻⁴ %
α-KGDH activity ≤3×10⁻⁴ %
GOT activity ≤5×10⁻⁴ %
GPT activity ≤5×10⁻⁴ %
Appearance: White powder, lyophilized
Additive: Dextran
Characteristics
1. Molecular weight: 64,000 (Gel filtration)
2. Isoelectric point: 4.3
3. Kₘ: 6.7×10⁻⁴ M (Pyruvate); 7.6×10⁻⁴ M (NADH)
4. Optimum pH: 5.0
5. pH stability: 5.5–8.5 (30°C, 1 hr)
6. Optimum temperature: 40°C
7. Thermal stability: up to 35°C (pH 7.0, 1 hr)
8. Application: Used for the enzymatic determination of numerous metabolites, e.g. ATP, ADP, glucose, creatinine, pyruvate, lactate and glycerol, and enzyme activities, e.g. GPT, PK and CPK by coupling with the related enzymes in clinical diagnosis.
Safe Handling
1. Do not inhale.
2. In case of direct contact with skin or eyes, immediately wash or rinse with plenty of water.
Assay method of Lactate dehydrogenase activity
Principle
The consumption of NADH is measured at 340 nm by spectrophotometry.
Unit Definition
One unit is defined as the enzyme quantity which oxidizes 1 μmol of NADH per minute under the conditions described below.
Reagents
A. 0.1M Tris-HCl buffer (pH 7.8)
B. 0.01 M Phosphate buffer (KH₂PO₄-NaOH (pH 7.0))
C. Substrate solution
Weigh 12.4 mg of β-NADH, add 20.5 mg of sodium pyruvate, then dissolve in 0.1M Tris-HCl buffer (A). Fill up to 100 ml with 0.1M Tris-HCl buffer (A). (Can be used for 5 days in light-shielded and refrigerated storage)
D. Enzyme solution
Weigh out Lactate Dehydrogenase (L139685) and dissolve in chilled 0.01M Phosphate buffer (B). Enzyme solution should be prepared so that the value of ΔOD/minute becomes in the range of 0.015±0.005/min.
Procedure
Pipette 2.9 mL of Substrate solution (C) into a quartz cell (d=10 mm) and keep at 25±0.5 °C for 5 minutes. Then, pipette 0.1 mL of Enzyme solution (D) into the quartz cell and mix well immediately. Keep the reaction mixture at 25±0.5 °C. Exactly at 2 minutes and 5 minutes after the addition of Enzyme Solution (D), measure the absorbances of the reaction mixture at 340 nm. (A2 and A5). As a blank, pipette 0.01M Phosphate buffer (B) into another quartz cell (d=10 mm) instead of Enzyme Solution (D), and take the same procedure described above. (Ab2 and Ab5)
Calculation
Lactate dehydrogenase activity (U/mg) = [(A2−A5)−(Ab2−Ab5)] / 3 × 1 / 6.22 × 3.0 × n / 0.1
3: Reaction time
6.22: Millimolar absorption coefficient of NADH at 340 nm
3.0: Volume of the reaction mixture
0.1: Volume of Enzyme solution
n: Dilution factor of Enzyme solution
K2605142
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