计算溶液所需的质量、体积或浓度。
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在色谱分析、有机合成和交叉偶联反应领域已被 2 篇同行评审文献引用。
Lactate Dehydrogenase (LDH) 是一种以 NAD⁺或 NADP⁺为受体、作用于供体 CH-OH 基团上的氧化还原酶,可以催化 D-乳酸氧化生成丙酮酸。D-Lactate dehydrogenase 广泛存在于细菌和真菌中,常用于生化研究。
用途
用于谷草转氨酶,谷丙转氨酶等试剂的研发和大量配制。
酶学性质
保护剂:BSA





活性测定方法
原理

反应消耗的NADH可用分光光度计在 340 nm检测。
酶活定义
单位酶活定义为在下述条件下,每分钟氧化1 μmol NADH 所需的酶量。
试剂准备
试剂 I:称取0.04 g丙酮酸钠,用0.1 M, pH 7.7 Tris-HCl缓冲液溶解并定容至100 mL。
试剂 II:称取2.11 g CAPS,用80 mL水溶解, 用NaOH调节pH至9.7,加入120 mg NADH, 用双蒸水定容至100 mL。
酶稀释液:10 mM, pH 7.0磷酸钾缓冲液,并 加入BSA至终浓度0.1%。
样品:用预冷的酶稀释液将酶稀释至1-2 U/mL。
操作步骤
4.1 向1 mL比色皿中加入1 mL试剂I,20μL待 测样品,混匀。
4.2 将反应混合物在37℃预热5 min。
4.3 向反应混合物中加入0.2 mL试剂 II,混匀,37℃反应,并用分光光度计在340 nm下,记录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(ΔAb)
∆A=∆As-∆Ab
活力计算

1.22:反应液总体积(mL);
0.02:酶液体积(mL);
1.0:光路长度 (cm);
df:稀释倍数;
C:酶浓度( mg/mL);
6.22:标准反应条件下,生色基团在340 nm处毫摩尔吸光系数(cm 2/μmol)。
Lactate Dehydrogenase (LDH) is an oxidoreductase that uses NAD⁺ or NADP⁺ as an acceptor and acts on the donor CH-OH group, and can catalyze the oxidation of D-lactate to pyruvate. D-Lactate dehydrogenase widely exists in bacteria and fungi, and is often used in biochemical research.
Application:
Used in the research and development and large-scale formulation of reagents such as glutamate transaminase.
Enzymatic Properties
Source: Microorganism
Classification: EC 1.1.1.28
Molecular Weight: 45 kDa (SDS-PAGE)
Isoelectric Point: 4.5
Km Value: 2.2×10⁻⁶ M (Pyruvate)
Inhibitors: Ag⁺, Hg²⁺
Optimal pH: 6.5-7.0 (Fig.1)
Optimal Temperature: 45℃ (Fig.2)
pH Stability: 5.5-10.0 (25℃, 16 h) (Fig.3)
Thermal Stability: Stable below 50℃ (pH 7.0, 30 min) (Fig.4)
Stability: Store at -25~-15℃ in the dark, maintain over 90% activity for 12 months (Fig.5)
Protectant: BSA





Assay Method
Principle

The NADH consumed in the reaction can be detected at 340 nm using a spectrophotometer.
Enzyme Activity Definition
One unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 μmol of NADH per minute under the following conditions.
Reagent Preparation
Reagent I: Weigh 0.04 g of sodium pyruvate, dissolve it in 0.1 M Tris-HCl buffer (pH 7.7), and dilute to 100 mL.
Reagent II: Weigh 21.1 g of CAPS, dissolve it in 80 mL of water, adjust the pH to 9.7 with NaOH, add 120 mg of NADH, and dilute to 100 mL with double-distilled water.
Enzyme Dilution Buffer: 10 mM potassium phosphate buffer (pH 7.0) with BSA added to a final concentration of 0.1%.
Sample: Dilute the enzyme with pre-cooled enzyme dilution buffer to 1-2 U/mL.
Operating Procedures
1. Operating Procedures4.1 Add 1 mL of Reagent I and 20 μL of the sample to be tested into a 1 mL cuvette, and mix well.
2. Preheat the reaction mixture at 37℃ for 5 min.
3. Add 0.2 mL of Reagent II to the reaction mixture, mix well, react at 37℃, and record the absorbance change (ΔAs) within 1 min at 340 nm using a spectrophotometer.
* Replace the enzyme with enzyme dilution buffer, and follow the same steps. The absorbance of the resulting solution is the blank absorbance (ΔAb).
∆A=∆As-∆Ab
Calculation

1.22: Total volume of the reaction solution (mL);
0.02: Volume of the enzyme solution (mL);
1.0: Path length (cm);
df: Dilution factor;
C: Enzyme concentration (mg/mL);
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| 批号(Lot Number) | 证书类型 | 货号 |
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| 分析证书 | D649636 | |
| 分析证书 | D649636 | |
| 分析证书 | D649636 | |
| 分析证书 | D649636 | |
| 分析证书 | D649636 | |
| 分析证书 | D649636 | |
| 分析证书 | D649636 |
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