Lactate Dehydrogenase (LDH-E) 60203 ラクテートデヒドロゲナーゼ(LDH-E)

  • 臨床診断用酵素
ラクテートデヒドロゲナーゼ(LDH-E)

臨床検査および連続乳酸測定センサにおいて、乳酸の測定に利用されます。

由来 recombinant E. coli
系統名

(s)-Lactate : ferricytochrome-c 2-oxidoreductase
EC 番号 1.1.2.3
反応式

L-Lactate + acceptor → → →　Pyruvate + reduced acceptor

仕様

Appearance Reddish brown lyophilizate
Activity ≧230 U/mg lyophilizate
Storage condition below -20℃ protected from light

特性

Molecular weight ca. 55 kDa (SDS-PAGE)
Structure tetramer
Coenzymes (Cofactors) FMN, Heme b (protoheme IX)
Michaelis constant 1.7×10-3M (L-Lactate)
pH Optimum 7.5
pH Stability 7.0–8.0
Optimum temperature 40℃
Thermal stability (liquid form) below 40℃

アプリケーション

The enzyme is useful for the determination of lactate in clinical analysis and continuous lactate monitoring sensor.

分析手順

Principle

The disappearance of the blue color of DCIP by the reduction is measured spectrophotometrically at 520 nm.

Definition of unit

One unit (U) causes the reduction of one micromole of DCIP per minute under the conditions described below.

Reagents

  1. L-Lactate solution, 50 mM: 0.56 g of sodium l-lactate/100 mL of distilled water.
  2. Potassium phosphate buffer, 1 M: pH 7.5: mix 1 M KH2PO4 solution and 1 M K2HPO4 solution to make a pH 7.5 solution.
  3. 2, 6-Dichloroindophenol (DCIP) solution, 1.8 mM: 58.7 mg of DCIP/100 mL of distilled water.
  4. 5-Methylphenazinium methyl sulfate (PMS) solution, 30 mM: 91.9 mg of PMS/10 mL of distilled water.
  5. Enzyme dilution buffer: 10 mM potassium phosphate buffer, pH 7.0, containing 0.15% bovine serum albumin(BSA).

Sample: dissolve the lyophilized enzyme to final concentration about 0.4 μg/mL with enzyme dilution buffer(Reagent E) immediately before measurement.

Procedure

  1. Pipette the following reagents into a cuvette (light path: 1 cm).
    600 μL l-Lactate solution (Reagent A)
    340 μL Potassium phosphate buffer pH 7.5 (Reagent B)
    500 μL DCIP solution (Reagent C)
    1360 μL Distilled water
  2. Equilibrate at 37℃ for about 3 min.
  3. Add 0.1 mL of PMS solution (Reagent D) and mix.
  4. Add 0.1 mL of sample and mix.
  5. Record the decrease of absorbance at 520 nm against water for 1 min. (30–90 sec) in a spectrophotometer thermostated at 37℃, and calculate the ΔA per min using the linear portion of the curve (ΔAS).
    The blank solution is prepared by adding Enzyme dilution buffer (Reagent E) instead of sample (ΔA0).

Calculation

Activity can be calculated by using the following formula:

6.8 : Millimolar extinction coefficient of DCIP under the assay condition (cm2/μmol)
1.0 : Light pass length (cm)
df : Dilution factor

実験データ

FAQ

LDH-Eの主な用途は何ですか？
LDH-Eの分子量を教えてください。
LDH-Eの至適pHと至適温度について教えてください。
LDH-Eの熱安定性について教えてください。
LDH-Eの保存方法を教えてください。
開封後の取扱い方法に注意事項はありますか？

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