G6PDH (Glucose-6-phosphate Dehydrogenase)
This enzyme is useful for enzymatic determination of glucose or ATP when coupled with hexokinase.
Specification
|
Appearance |
White amorphous powder, lyophilized |
|
Purity(SDS-PAGE) |
≥90.0% |
|
Enzyme powder specific activity |
≥420.0 U/mg |
|
NADH/NADPH oxidase |
≤0. 1% |
|
Hexokinase |
≤0.001% |
|
Glutathione reductase |
≤0.001% |
|
Phosphoglucose isomerase |
≤0.001% |
Properties
|
EC number |
1.1.1.49 (Recombinant from microorganism) |
|
|
Molecular weight |
57 kDa (SDS-PAGE) |
|
|
Isoelectric point |
4.6 |
|
|
Michaelis constants |
3.2× 10-4 M (NAD+), 2.6× 10-4 M (G-6-P) |
|
|
Inhibitors |
SDS, Zn2+, Fe3+ |
|
|
Optimum pH |
8.0 |
Fig. 1 |
|
Optimum temperature |
45 ℃ |
Fig. 2 |
|
pH stability |
pH 5.5 – 10.0 (25 ℃, 16h) |
Fig. 3 |
|
Thermal stability |
Below 40 ℃ (pH 7.5, 30 min) |
Fig. 4 |
|
Storage stability |
At least one year at -25 ～- 15 ℃ |
Fig. 5 |
|
Stabilizers |
BSA |
Figures
Assay Principle
The appearance of NADH is measured at 340nm by spectrophotometry.
Unit definition
One unit (U) is defined as the amount of enzyme which produces 1 μmol of NADH per min under the conditions described below.
Reagents preparation
Reagent I: 55 mM Tris-HCl, pH 7.8, contains 3.3 mM MgCl2
Reagent II: 60 mM NAD+
Reagent III: 0. 1 M D-Glucose-6-phosphate solution.
Enzyme diluent: 5 mM pH 7.5 Tris HCl, contains 0. 1% BSA.
Sample: Dilute the sample to 0.15-0.25 U/mL by Enzyme diluent.
Procedure
1. Add 2.7 mL Reagent I, 0. 1 mL Reagent II and 0. 1 mL Reagent III to the 3mL cuvette and preincubate at 37 ℃ for 5 min.
2. Add 0. 1mL the diluted enzyme solution and mix.
3. Record the ΔAs at 340 nm in 1 minute in a spectrophotometer thermostated at 37 ℃ .
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As - ∆Ab
Calculation
3.00: Total volume (mL)
0. 10: Enzyme volume (mL)
1.0: Light path length (cm)
df: dilution factor
C: Enzyme concentration (mg/mL)
6.22: Millimolar extinction coefficient of NADH under 340 nm (cm2/μmol)
References
1. Haberstich, H. V. and Zuber, H.（1971）Arch. Biochem. Biophys., 144, 245–252.
2. Muramatsu, N.（ 1974）Arch. Microbiol., 98, 275–289.
3. Ishaque, A., Milhausen, M., Levy, H. R.（1974） Biochem. Biophys. Res. Commun., 59, 894–901.
4. Milhausen, M. and Levy, H. R.（1975）Eur. J. Biochem., 50, 453–461.
