DIAPHORASE from Microorganism
Appearance | Yellowish amorphous powder, lyophilized | |
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Activity | GradeⅢ 500 U/mg-solid or more | |
Contaminants | Myokinase | ≤ 5.0×10-1 % |
NADH oxidase | ≤ 1.0×10-1 % |
Stability | Stable at −20 ℃(Fig.1) |
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Molecular weight(Gel-filtration) | approx. 48,000 |
Michaelis constant | 2.2×10-4 M(NADH),2.9×10-2 M(NADPH) |
Inhibitors | Fe3+, Mn2+, Cu2+, Pb2+ |
Isoelectric point | 5.0 |
Optimum pH | 8.0(Fig.3) |
Optimum temperature | 60 ℃(Fig.4) |
pH Stability | 5.0−10.0(Fig.5) |
Thermal stability | below 70 ℃(Fig.6) |
Substrate specificty | Either NADH or NADPH can be used as a reductant. |
Effect of various chemicals | (Table 1) |
This enzyme is useful for colorimetric determination of NAD(P)H and many dehydrogenases in combination with various dyes that act as hydrogen acceptors from NAD(P)H.
Reduction of 2,6-dichlorophenol-indophenol (DCPIP) is measured at 600 nm by spectrophotometry.
One unit causes decrease in DCPIP by one unit of absorbance (1.0) per minute under the conditions detailed below.
A. Buffer solution | 0.2 M Tris-HCl, pH 8.0 |
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B. NADH solution | 36 mM (Prepare freshly and store on ice) |
C. DCPIP solution | 2.4 mM 7.8 mg of DCPIP (MW: 326.11) / 10 mL of H2O; should be prepared fresh |
D. Enzyme diluent | Buffer solution (A) containing 0.5 % of Tween 20 |
1.Prepare the following reaction mixture in a cuvette (d = 1.0 cm) and equilibrate at 37 ℃ for approximately 4 minutes.
2.4 mL | H2O | |
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0.3 mL | Buffer solution | (A) |
0.1 mL | NADH solution | (B) |
Concentration in assay mixture | |
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Tris buffer | 27 mM |
NADH | 1.2 mM |
DCPIP | 80 μM |
Tween20 | ca.167 μg/mL |
2.Add 0.1 mL of the enzyme solution* and mix by gentle pipetting and equilibrate at 37 ℃ for another 1 min.
3.Add 0.1 mL of DCPIP solution (C) and mix by rapid inversion.
4.Record the decrease in optical density at 600 nm against water for 3 to 4 min with a spectrophotometer thermostated at 37 ℃, and calculate the ΔOD per minute from the initial linear portion of the curve (OD test).
At the same time, measure the blank rate (OD blank) by 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 0.10−0.25 U/mL with ice-cold enzyme diluent (D) and store on ice.
Activity can be calculated by using the following formula :
Volume activity (U/mL) =
ΔOD/min (OD test−OD blank)×Vt×df
20.9×1.0×Vs
= ΔOD/min×1.43×df
Weight activity (U/mg) = (U/mL)×1/C
Vt | : Total volume (3.0 mL) |
Vs | : Sample volume (0.1 mL) |
20.9 | : Millimolar extinction coefficient of DCPIP under the assay conditions (cm2/micromole) |
1.0 | : Light path length (cm) |
df | : Dilution factor |
C | : Enzyme concentration in dissolution (c mg/mL) |
[The enzyme dissolved in 0.1 M HEPES buffer, pH 7.5 (5 U/mL) was incubated with each chemical at 25 ℃ for 1 hr.]
Chemical | Concn.(mM) | Residual activity(%) |
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None | ー | 100 |
Metal salt | 2.0 | |
MgCl2 | 102 | |
CaCl2 | 99 | |
Ba(OAc)2 | 100 | |
FeCl3 | 4.4 | |
CoCl2 | 94 | |
MnCl2 | 55 | |
ZnCl2 | 84 | |
Cd(OAc)2 | 101 | |
NiCl2 | 101 | |
CuSO4 | 23 | |
Pb(OAc)2 | 46 | |
AgNO3 | 94 | |
MIA | 1.0 | 104 |
NaF | 2.0 | 105 |
Chemical | Concn.(mM) | Residual activity(%) |
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NaN3 | 2.0 | 104 |
EDTA | 5.0 | 105 |
o-Phenanthroline | 2.0 | 105 |
α,α′-Dipyridyl | 1.0 | 102 |
Borate | 5.0 | 104 |
IAA | 2.0 | 105 |
NEM | 2.0 | 106 |
Hydroxylamine | 2.0 | 107 |
TritonX-100 | 0.10 % | 109 |
Brij 35 | 0.10 % | 109 |
Tween 20 | 0.10 % | 116 |
Span 20 | 0.10 % | 113 |
Na-Cholate | 0.10 % | 110 |
SDS | 0.05 % | 91 |
DAC | 0.05 % | 110 |
Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. Stability (Powder form)
(Kept under dry conditions)
Fig.3. pH-Activity
37 ℃, in 0.1 M buffer solution;〇――〇, KPB; △――△, Tris-HCl;□――□, Gly-NaOH
Fig.4. pH-Stability
25 ℃, 20 hr-treatment with 0.1 M buffer solution; 〇――〇, Glycine-HCl;▲――▲, Acetate; □――□, KPB;●――●, Tris-HCl; ×――×, Glycine-NaOH
Fig.5. Temperature activity
(in 50 mM K-Phoshate buffer, pH 7.5)
Fig.6. Thermal stability
15 min-treatment with 0.1 M K-Phosphate buffer, pH 7.5 Enzyme concentration : 10 U/mL
1. 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
酵素溶液:酵素標品を予め氷冷した蒸留水で溶解し,分析直前に酵素希釈液(D)で0.10〜0.25U/mLに希釈する。
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。
2.4 mL | 蒸留水 | |
0.3 mL | 0.2M Tris-HCl緩衝液、pH 8.0 | (試薬A) |
0.1 mL | 36mM NADH水溶液 | (試薬B) |
2.酵素溶液0.1 mLを加えてピペッティングによる混和後,さらに約1分間予備加温する。
3.DCPIP水溶液(試薬C)を0.1 mLを加えて,速やかに転倒混和した後,水を対照に37℃に制御された分光光度計で600nmの吸光度変化を3〜4分間記録し,その初期直線部分より1分間当たりの吸光度変化を求める(ODtest)。
4.盲検は1の反応混液に酵素希釈液(0.5 %のTween20を含む試薬A),DCPIP水溶液各0.1 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量を求める(ODblank)。
5.計算式
U/mL =
ΔOD/min (OD test−OD blank)×3.0(mL)×希釈倍率
20.9×1.0×0.10(mL)
= ΔOD/min×1.43×希釈倍率 | |
U/mg | =U/mL×1/C |
20.9 | : DCPIP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) |
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