GLUCOSE-6-PHOSPHATE DEHYDROGENASE from Microorganism
Appearance | White amorphous powder, lyophilized | |
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Activity | GradeⅢ 200 U/mg-solid or more | |
Contaminants | Creatine phosphokinase | ≤ 1×10-3 % |
Phosphoglucomutase | ≤ 1×10-3 % | |
6-Phosphogluconate dehydrogenase | ≤ 5×10-3 % | |
Phosphoglucose isomerase | ≤ 1×10-2 % | |
Glutathione reductase | ≤ 1×10-3 % | |
Hexokinase | ≤ 1×10-2 % | |
Myokinase | ≤ 1×10-2 % | |
NADH oxidase | ≤ 1×10-2 % | |
NADPH oxidase | ≤ 1×10-2 % |
Stability | Stable −20 ℃ for at least one year(Fig.1) | |
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Molecular weight | approx. 140,000 (by gel filtration) | |
Michaelis constants | NAD+ linked | 2.4×10-4 M (NAD+), 4.7×10-4 M (G-6-P) |
NADP+ linked | 7.4×10-6 M (NADP+), 3.2×10-4 M (G-6-P) | |
Inhibitors | Metal ions, iodoacetamimide, SDS etc. | |
Optimum pH | 7.8(Fig.2) | |
Optimum temperature | 50−55 ℃(Fig.3) | |
pH Stability | pH 5.0−11.0 (25 ℃, 22 hr)(Fig.4) | |
Thermal stability | below 50 ℃ (pH 7.8, 30 min)(Fig.5) | |
Substrate specificity | (Table 1) | |
Effect of various chemicals | (Table 2) |
This enzyme is useful for enzymatic determination of NAD+(NADP+) and G-6-P, and the activities of phosphoglucose isomerase, phosphoglucomutase and hexokinase. The enzyme is also used for enzymatic determination of glucose and creatine phosphokinase activity in combination with hexokinase (HXK-311).
The formation of NADH is measured at 340 nm by spectrophotometry.
One unit causes the formation of one micromole of NADH per minute under the conditions detailed below.
A. Tris-HCl buffer, pH 7.8 | 55 mM (containing 3.3 mM magnesium chloride) | |
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B. NAD+ solution | 60 mM (Should be prepared fresh) | |
C. G-6-P solution | 0.1 M D-Glucose-6-phosphate (should be prepared fresh) | |
D. Enzyme diluent | 5 mM Tris-HCl buffer, pH 7.5, containing 0.1 % bovine serum albumin. |
1. Prepare the following reaction mixture in a cuvette (d = 1.0 cm) and equilibrate at 30 ℃ for about 5 minutes.
2.7 mL | Tris-HCl buffer, pH 7.8 | (A) |
0.1 mL | NAD+ solution | (B) |
0.1 mL | G-6-P solution, pH 7.8 | (C) |
Concentration in assay mixture | |
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Tris-HCl buffer | 50 mM |
G-6-P | 3.3 mM |
NAD+ | 2.0 mM |
MgCl2 | 3.0 mM |
BSA | 33 μg/mL |
2. Add 0.1 mL of the enzyme solution* and mix by gentle inversion.
3. Record the increase of optical density at 340 nm against water for 4 to 5 minutes with a spectrophotometer thermostated at 30 ℃, 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) the same method as the test except that the enzyme diluent (D) is added instead of the enzyme solution.
*Dissolve the enzyme preparation in ice-cold enzyme diluent (D) and dilute to 0.05−0.20 U/mL with the same buffer, immediately before the assay.
Activity can be calculated by using the following formula :
Volume activity (U/mL) =
ΔOD/min (OD test−OD blank)×Vt×df
6.22×1.0×Vs
= ΔOD/min×4.82×df
Weight activity (U/mg) = (U/mL)×1/C
Vt | : Total volume (3.0 mL) |
Vs | : Sample volume (0.1 mL) |
6.22 | : Millimolar extinction coefficient of NADH under the assay condition (cm2/micromole) |
1.0 | : Light path length (cm) |
df | : Dilution facter |
C | : Enzyme concentration in dissolution |
[3.3 mM of substrate, 50 mM Tris-HCl buffer, pH 7.8]
Substrate | Relative activity(%) |
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Glucose-6-phosphate | 100 |
Fructose-6-phosphate | 0 |
Glucose-1-phosphate | 0 |
Gluconate-6-phosphate | 0 |
[The enzyme dissolved in 50 mM Tris-HCl buffer, pH 7.5 (10 U/mL) was incubated with each chemical for 1 hr at 30 ℃.]
Chemical | Concn.(mM) | Residual activity(%) |
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None | - | 100 |
Metal salt | 2.0 | |
AgNO3 | 74 | |
Ba(OAc)2 | 100 | |
CaCl2 | 95 | |
Ca(OAc)2 | 84 | |
CoCl2 | 94 | |
CuSO4 | 84 | |
FeCl3 | 0 | |
FeSO4 | 0 | |
HgCl2 | 87 | |
MgCl2 | 100 | |
MnCl2 | 97 | |
NiCl2 | 94 | |
Pb(OAc)2 | 31 | |
Zn(OAc)2 | 63 | |
ZnSO4 | 76 | |
KF | 2.0 | 103 |
NaF | 2.0 | 99 |
NaN3 | 2.0 | 102 |
Chemical | Concn.(mM) | Residual activity(%) |
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NEM | 2.0 | 94 |
PCMB | 2.0 | 103 |
MIA | 2.0 | 99 |
Iodoacetamide | 2.0 | 0 |
EDTA | 5.0 | 102 |
(NH4)2SO4 | 20.0 | 99 |
Borate | 20.0 | 98 |
o-Phenanthroline | 2.0 | 100 |
α,α′-Dipyridyl | 2.0 | 102 |
Urea | 2.0 | 99 |
Guanidine | 2.0 | 100 |
Hydroxylamine | 2.0 | 100 |
Na-cholate | 1.0 % | 106 |
Triton X-100 | 1.0 % | 104 |
Brij 35 | 1.0 % | 12 |
SDS | 0.1 % | 1 |
Tween 20 | 0.1 % | 102 |
Span 20 | 0.1 % | 101 |
DAC | 0.1 % | 1 |
Ac, CH3CO; NEM, N-Ethylmaleimide; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammoniumchloride.
Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. pH-Activity
30 ℃ in the following buffer solution: pH 6.0-7.0, 50 mM PIPES pH 7.2-9.0, 50 mM Tris-HCI
Fig.3. Temperature activity
(in 50 mM Tris-HCI buffer, pH 7.8)
Fig.4. pH-Stability
25 ℃, 22 hr-treatment with the following 0.1 M buffer solution: pH 5.0-6.0,Acetate; pH 6.0-9.0,K-phosphate; pH 9.0-11.0,Glycine-NaOH
Fig.5. Thermal stability
30 min-treatment with 5.0 mM Tris-HCI buffer, pH 7.8, containing 0.1 % of bovine serum albumin
1. 原理
生成したNADHの生成量を340nmにおける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を生成する酵素量を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0.1 %牛血清アルブミン(BSA)を含む5mM Tris-HCl緩衝液,pH7.5で溶解し,分析直前に同緩衝液で0.05〜0.20U/mLに希釈する。
4.手順
1.下記反応混液をキュベット(d=1.0cm)に調製し,30℃で約5分間予備加温する。
2.7 mL | Tris-HCl 緩衝液 | (A) |
0.1 mL | NAD+水溶液 | (B) |
0.1 mL | 基質溶液 | (C) |
2.酵素溶液0.1 mLを添加し,ゆるやかに混和後,水を対照に30℃に制御された分光光度計で340nmの吸光度変化を4〜5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(ΔODtest)。
3.盲検は酵素溶液の代りに酵素希釈液(0.1 %BSAを含む5mM Tris-HCl緩衝液,pH7.5)を0.1 mL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。
5.計算式
U/mL =
ΔOD/min (OD test−OD blank)×3.0(mL)×希釈倍率
6.22×1.0×0.1(mL)
= ΔOD/min×4.82×希釈倍率 | |
U/mg | = U/mL×1/C |
6.22 | : 上記測定条件における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) |
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