TOYOBO Ideas & Chemistry Biotechnology Operating
Department

PREPARATION and SPECIFICATION

Appearance White amorphos powder, lyophilized
Activity GradeⅡ　400 U/mg-solid or more
Contaminants NADH oxidase ≤1.0 × 10-3 %
Malate dehydrogenase ≤1.0 × 10-2 %
GOT ≤5.0 × 10-3 %
GPT ≤5.0 × 10-3 %
Myokinase ≤1.0 × 10-2 %
Pyruvate kinase ≤1.0 × 10-3 %

PROPERTIES

Stability Stable at －20 ℃
Molecular weight approx. 140,000
Isoelectric point 4.0
Michaelis constants 1.6 × 10-4 M (pyruvate, pH 7.0)
Inhibitors Ag＋, Hg2＋, SH-reagents
Optimum pH 6.0－7.0(Fig.2)
Optimum temperature 35－40 ℃(Fig.3)
pH Stability pH 5.0－9.0 (25 ℃, 48 hr)(Fig.4)
Thermal stability below 45 ℃ (pH 7.0, 15 min)(Fig.5)
Effect of various chemicals (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of numerous metabolites, such as ATP, ADP, glucose, creatinine, pyruvate, lactate and glycerol, and the activities of enzymes such as GPT, PK and CPK, in combination with related enzymes.

ASSAY

Principle

The formation of NADH is measured at 340 nm by spectrophotometry.

Unit definition

One unit causes the oxidation of one micromole of NADH per minute under the conditions detailed below.

Method

Reagents

A. Pyruvate solution 5.0 mM［5.50 mg sodium pyruvate (MW＝110)/0.35 mL of H2O］(Should be prepared fresh)
B. K-Phosphate buffer, pH 7.4 1.0 M
C. NADH solution 1.0 mM［7.63 mg NADH・2 Na (MW＝763)/0.35 mL of H2O］(Should be prepared fresh)
D. Enzyme diluent 0.1 M K-phosphate buffer, pH 7.4 contg. 0.1 % of BSA

Procedure

1. Prepare the following working solution (for 10 tests) in a brownish bottle, immediately before use and store on ice.

3.0 mL Substrate solution (A)
0.35 mL Potassium phosphate, pH 7.4 (B)
0.35 mL NADH solution (C)
0.35 mL H2O (D)
Concentration in assay mixture
Potassium phosphate buffer 67 mM
Pyruvate 0.49 mM
NADH 0.098 mM
BSA 16.4 μg/mM

2. Pipette 0.35 mL of working solution into a cuvette (d = 1.0 cm) and equilibrate at 25 ℃ for approximately 5 minutes.

3. Add 0.35 mL of the enzyme solution* and mix by gentle inversion.

4. Record the decrease in optical density at 340 nm against water for 2 to 3 minutes with a spectrophotometer thermostated at 25℃, and calculate the ΔΔOD per minute from the initial linear portion of the curve (ΔOD test).

At the same time, measure the blank rate (ΔΔOD) using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

*Dilute the enzyme preparation to 0.35 mL with ice-cold enzyme diluent (D), immediately before the assay.

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/mL) =

  • ΔOD/min (ΔOD test－ΔOD blank)×Vt×df

    6.22×1.0×Vs

  • ＝ ΔOD/min×9.81×df

Weight activity (U/mg) ＝ (U/mL)×1/C

Vt : Total volume (0.35 mL)
Vs : Sample volume (0.35 mL)
6.22 : Millimolar extinction coefficient of NADH (cm2/micromole)
1.0 : Light path length (cm)
df : Dilution factor
C : Enzyme concentration in dissolution (c mg/mL)

REFERENCES

1) C.A.Loshon, R.B.McComb, L.W.Bond, G.N.Bowers, Jr.W.H.Coleman and R.H.Gwynn; Clin.Chem., 23, 1576 (1977).

2) H.Taguchi, M.Machida, H.Matsuzawa and T.Ohta; Agric.Biol.Chem., 49(2), 359 (1985).

3) F.Gasser, M.Doudoroff, and R.Contopoulos; J.Gen. Microbiol. 62, 241 (1970).

Table 1. Effect of Various Chemicals on D-Lactate dehydrogenase

[The enzyme dissolved in 0.1 M K-phosphate buffer, pH 7.4 (0.35 mL) was incubated with each chemical at 25 ℃ for 1 hr.]

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 2.0
    MgCl2 100
    CaCl2 96.3
    Ba(OAc)2 95.8
    FeCl3 94.4
    CoCl2 97.3
    MnCl2 96.9
    Cd(OAc)2 97.2
    NiCl2 95.5
    CuSO4 94.3
    Pb(OAc)2 96.0
    AgNO3 71.5
  • Chemical Concn.(mM) Residual
    activity(%)
    MIA 2.0 92.4
    NaF 2.0 98.0
    NaN3 20 97.2
    EDTA 5.0 96.0
    o-Phenanthroline 2.0 98.0
    α,α′-Dipyridyl 1.0 97.1
    Borate 50 97.5
    IAA 2.0 91.6
    NEM 2.0 95.2
    Hydroxylamine 2.0 96.3
    Triton X-100 0.10 % 105.5
    Brij 35 0.10 % 104.1
    Tween 20 0.10 % 106.3
    Span 20 0.10 % 98.2
    Na-cholate 0.10 % 102.1
    SDS 0.05 % 104.4
    DAC 0.05 % 47.6

Ac, CH3CO; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; NEM, N-ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

  • Fig.1. Stability (Powder form)

    Fig.1. Stability (Powder form)

    (kept under dry condition, 37 ℃)

  • Fig.2. pH-Activity

    Fig.2. pH-Activity

    in 57 mM buffer solution: pH 4-5, acetate; pH 5-8, K-phosphate; pH 8-9, Tris-HCl

  • Fig.3. Temperature activity

    Fig.3. Temperature activity

    (in 67 mM K-phosphate buffer, pH 7.4)

  • Fig.4. pH-Stability

    Fig.4. pH-Stability

    25 ℃, 48hr-treatment with 0.1 M buffer solution: pH 4-6, dimethylglutaric acid-NaOH; pH 6-8, K-phosphate; pH 8-9, Tris-HCl; pH 9-10, glycine-NaOH. Enzyme concentration: 0.35 mL

  • Fig.5. Temperature stability

    Fig.5. Temperature stability

    15 min-treatment with 50 mM K-phosphate buffer, pH 7.0. Enzyme concentration: 0.35 mL

活性測定法（Japanese）

1. 原理

NADHの消失量を340nmの吸光度の変化で測定する｡

2.定義

下記条件下で1分間に1マイクロモルのNADHが酸化される酵素量を1単位(U)とする。

3.試薬

  • 5.0mM ピルビン酸ナトリウム水溶液(用時調製)
  • 1.0M K-リン酸緩衝液，pH7.4
  • 1.0mM NADH水溶液(用時調製)
  • 酵素溶解液：0.1M K-リン酸緩衝液, pH 7.4
  • 酵素希釈液：0.1 % BSAを含む0.1M K-リン酸緩衝液, pH 7.4

酵素溶液：酵素標品を予め氷冷した酵素溶解液(D)で溶解し,分析直前に酵素希釈液(E)で0.2〜1.0U/mLlに希釈する。

4.手順

1.下記反応混液を使用直前に調製する。(褐色瓶にて氷冷保存)

3.0 mL 基質溶液 (試薬A)
2.0 mL K-リン酸緩衝液 (試薬B)
3.0 mL NADH水溶液 (試薬C)
22.0 mL H2O

2.反応混液3.0 mLをキュベット(d＝1.0cm)に採り,25℃で約5分間予備加温する。

3.酵素溶液0.05 mLを添加し,ゆるやかに混和後,水を対照に25℃に制御された分光光度計で340nmの吸光度変化を2〜3分間記録し,その初期直線部分から1分間当たりの吸光度変化を求める(ΔOD test)。

4.盲検は反応混液①3.0 mLに酵素溶液の代わりに酵素希釈液(E)0.05 mLを加え,上記同様に操作を行って1分間当たりの吸光度変化を求める(ΔODblank)。

5.計算式

  • U/mL =

  • ΔOD/min (ΔOD test－ΔOD blank)×3.05(mL)×希釈倍率

    6.22×1.0×0.05(mL)

＝ ΔOD/min×9.81×希釈倍率
U/mg = U/mL×1/C
6.22 : NADHのミリモル分子吸光係数(cm2/micromole)
1.0 : 光路長(cm)
C : 溶解時の酵素濃度(c mg/mL)