PREPARATION and SPECIFICATION

Appearance White amorphous 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 ℃ for at least one year(Fig.1)
Molecular weight approx. 140,000 (by gel filtration)
Isoelectric point 4.0
Michaelis constant 1.6×10-4 M (pyruvate, pH 7.0)
Inhibitors Ag, Hg2+, SH-reagents
Optimum pH 6.0−7.0(Fig.3)
Optimum temperature 35−40 ℃(Fig.4)
pH Stability pH 5.0−9.0 (25 ℃, 48 hr)(Fig.5)
Thermal stability below 45 ℃ (pH 7.0, 15 min)(Fig.6)
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)/10 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・2Na (MW=763)/10 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)
2.0 mL Potassium phosphate buffer, pH 7.4 (B)
3.0 mL NADH solution (C)
22.0 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 3.0 mL of working solution into a cuvette (d = 1.0 cm) and equilibrate at 25 ℃ for approximately 5 minutes.

3. Add 0.05 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 ΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

*Dilute the enzyme preparation to 0.2−1.0 U/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 (3.05 mL)
Vs : Sample volume (0.05 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 (20 U/mL) was incubated with each chemical at 25 ℃ for 1 hr.]

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 2.0
    MgCl2 93.0
    CaCl2 99.8
    Ba(OAc)2 98.1
    FeCl2 87.9
    CoCl2 91.5
    MnCl2 92.2
    ZnSO4 89.6
    Cd(OAc)2 91.3
    NiCl2 92.6
    CuSO4 93.8
    Pb(OAc)2 93.2
    AgNO3 73.0
    HgCl2 0
  • Chemical Concn.(mM) Residual
    activity(%)
    PCMB 2.0 89.3
    MIA 2.0 0.1
    NaF 2.0 98.3
    NaN3 20 94.6
    EDTA 5.0 99.2
    o-Phenanthroline 2.0 95.3
    α,α′-Dipyridyl 1.0 93.7
    Borate 50 95.6
    IAA 2.0 33.6
    NEM 2.0 97.7
    Hydroxylamine 2.0 95.6
    Triton X-100 0.10 % 121
    Brij 35 0.10 % 116
    Tween 20 0.10 % 117
    Span 20 0.10 % 105
    Na-cholate 0.10 % 112
    SDS 0.05 % 109
    DAC 0.05 % 52.0

Ac, CH3CO; PCMB, p-Chloromercuribenzoate; 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 conditions)

  • Fig.2. Stability (Powder form)

    Fig.2. Stability (Powder form)

    (kept under dry conditions)

  • Fig.3. pH-Activity

    Fig.3. pH-Activity

    in 67 mM buffer solution; pH 4-5, acetate;pH 5-8,K-phosphate;pH 8-9 Tris-HCI

  • Fig.4. Temperature activity

    Fig.4. Temperature activity

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

  • Fig.5. pH-Stability

    Fig.5. pH-Stability

    25 ℃,48hr-treatment with 0.1 M buffer solution: pH 4-6, dimethylgutaric acid-NaOH;pH 6-8, K-phosphate; pH 8-9,Tris-HCI; pH 9-10,glycine-NaOH. enzyme concn.: 10 U/mL

  • Fig.6. Temperature stability

    Fig.6. Temperature stability

    15 min-treatment with 50 mM K-phosphate buffer,pH 7.0.enzyme concn.: 10 U/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)

酵素溶液:分析直前に酵素標品を予め氷冷した0.1%BSAを含む0.1M K-リン酸緩衝液,pH7.4で溶解(約1mg/ml)し,分析直前に同緩衝液で0.2〜1.0U/ml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)

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

2.反応混液3.0ml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0.05ml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 当りの吸光度変化を求める(ΔOD test)。

4.盲検は反応混液①3.0mlに酵素溶液の代りに酵素希釈液(0.1% BSAを含む0.1M K-リン酸緩衝液, pH7.4)を0.05mlを加え,上記同様に操作を行って,1分間当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)