PREPARATION and SPECIFICATION

Appearance Yellowish amorphous powder, lyophilized
Activity GradeⅢ 30U/mg-solid or more
(containing approx. 15% of stabilizers)
Contaminants Myokinase ≤5.0×10-1%
NAD(P)H oxidase  ≤5.0×10-1%
Stabilizers FMN, NAD(P)H

PROPERTIES

StabilityStable at −20℃ for at least one year(Fig.1)
Molecular weight24,000 1)
Michaelis constants2.0×10-5M(NADH),6.0×10-6M(NADPH)
StructureOne mol of FMN per mol of enzyme 1)
InhibitorN-Ethylmaleimide
Optimum pH8.5(Fig.3)
Optimum temperature50℃(Fig.4)
pH StabilitypH7.5(30℃, 3hr)(Fig.5)
Thermal stabilitybelow 30℃(pH 7.5, 30min)(Fig.6)
Substrate specifictyEither NADH or NADPH can be used as a reductant.
The catalytic ratio (NADPH/NADH) is 0.6 in the assay method.
Neither oxygen nor cytochrome C can be utilized as a hydrogen acceptor.

APPLICATIONS

This enzyme is useful for colorimetric determination of NAD(P)H and many dehydrogenases when coupled with various dyes which act as hydrogen acceptors from NAD(P)H.

ASSAY

Principle

Principle

The reduction of DCPIP(2,6-dichlorophenol-indophenol) is measured at 600 nm by spectrophotometry.

Unit definition

One unit causes the decrease of one unit absorbance (1.0) of DCPIP per minute under the condeitions described below.

Method

Reagents

A. Buffer solution 0.2M Tris-HCl, pH 7.5
B. NADH solution 6.0mM (Prepare freshly and store on ice)
C. DCPIP solution 1.2mM[3.9mg DCPIP・2H2O/10ml of H2O](Should be prepared fresh)
D. Enzyme diluent Buffer solution(A) containing 0.1% of bovine serum albumin.

Procedure

1.Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 25℃ for about 5 minutes.

2.4ml H2O
0.3ml Buffer solution (A)
0.1ml NADH solution (B)
Concentration in assay mixture
Tris buffer 27 mM
NADH 0.20 mM
DCPIP 40 μM
BSA ca.33μg/ml

2.Add 0.1ml each of the enzyme solution* and DCPIP solution (C) in this order and mix by rapid inversion.

3.Record the decrease of optical density at 600nm against water for 2 to 3 minutes in a spectrophotometer thermostated at 25℃, and calculate theΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

*Dissolve the enzyme preparation in ice-cold buffer solution (A) (approx.1.0% solution), dilute to 0.4−0.8U/ml with ice-cold enzyme diluent (D) and store on ice.

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/ml) =

  • ΔOD/min (ΔOD test−ΔOD blank)×df


    1.0×Vs

= ΔOD/min×10×df

Weight activity (U/mg) = (U/ml)×1/C

Vs : Sample volume (0.1ml)
1.0 : Unit absorbance at 600nm due to unit definition
df : Dilution factor
C : Enzyme concentration in dissolution (c mg/ml)

REFERENCES

1)F.Kaplan, P.Setlow and N.O.Kaplan; Arch,Biochem.Biophys., 132, 91 (1969).

Table 1. Effect of Various Chemicals on Diaphorase

[The enzyme dissolved in 0.2M Tris-HCl buffer, pH 7.5 (40U/ml) was incubated with each chemical at 25℃ for 1hr.]

  • Chemical Concn.(mM) Residual activity(%)
    None 100
    Metal salt 2.0
    MgCl2 99
    CaCl2 102
    Ba(OAc)2 100
    FeCl2 90
    CoCl2 101
    MnCl2 96
    ZnCl2 100
    Cd(OAc)2 100
    NiCl2 99
    CuSO4 87
    Pb(OAc)2 88
    AgNO3 103
    HgCl2 103
    PCMB 2.0 90
    MIA 1.0 100
  • Chemical Concn.(mM) Residual activity(%)
    NaF 2.0 102
    NaN3 2.0 100
    EDTA 5.0 99
    o-Phenanthroline 2.0 99
    α,α′-Dipyridyl 1.0 101
    Borate 5.0 100
    IAA 2.0 99
    NEM 2.0 100
    Hydroxylamine 2.0 101
    TritonX-100 0.10% 106
    Brij 35 0.10% 104
    Tween 20 0.10% 107
    Span 20 0.10% 101
    Na-Cholate 0.10% 99
    SDS 0.05% 32
    DAC 0.05% 32
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

    The enzyme reaction was carried out with NADH(◯)or NADPH(●).buffer used: pH6.0-7.5, 10mM Veronal-acetate;pH7.5-9.0, 33mM Tris-HCI;pH9.2-10.2, 33mM NH4OH-NH4OH

  • Fig.4. Temperature activity

    Fig.4. Temperature activity

    The enzme reaction was carried out with NADH(◯)or NADPH(●).
    20mM Tris-HCI buffer,pH8.5

  • Fig.5. pH-Stability

    Fig.5. pH-Stability

    30℃,3hr-treatment with the following buffer solution: pH6.0-7.5, 10mM Veronalacetate; pH7.5-9.0, 33mM Tris-HCI

  • Fig.6. Thermal stability

    Fig.6. Thermal stability

    30min-treatment with 0.2M Tris-HCI buffer,pH7.5 enzyme concentration:40U/ml

活性測定法(Japanese)

1. 原理

原理

DCPIP(2,6-dichlorophenol-indophenol)の還元量を600nm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600nmの吸光度を1.0減少させる酵素量を1単位(U)とする。

3.試薬

  • 0.2M Tris-HCl緩衝液, pH7.5
  • 6.0mM NADH水溶液(用時調製,氷冷保存)
  • 1.2mM DCPIP水溶液〔3.9mgのDCPIP・2H2O(MW=326.11)を10mlの蒸留水で溶解する〕(用時調製)

酵素溶液:酵素標品を予め氷冷した試薬Aに溶解し(約1.0%溶液),予め氷冷した0.1%牛血清アルブミン(BSA)を含む試薬Aで0.4〜0.8U/mlに希釈し,氷冷保存する。

4.手順

1.下記反応液をキュベット(d=1.0cm)に採り,25℃で約5分間予備加温する。

2.4ml 蒸留水 (A)
0.3ml Tris-HCl (B)
0.1ml NADH水溶液 (C)

2.酵素溶液,DCPIP水溶液(C)各0.1mlをこの順序で添加し,ゆるやかにかつ速やかに混和後,水を対照に25℃に制御された分光光度計で600nmの吸光度変化を2〜3分間記録し,その初期直線部分より1分間当りの吸光度変化を求める(ΔODtest)。

3.盲検は反応混液1に酵素希釈液(0.1%BSAを含む試薬A),DCPIP水溶液(C)各0.1ml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)×希釈倍率


    1.0×0.1(ml)

= ΔOD/min×10×希釈倍率
U/mg =U/ml×1/C
1.0 : 活性定義に基いて定められた600nmにおける単位吸光度
C : 溶解時の酵素濃度(c mg/ml)