HK (Hexokinase)
This enzyme is useful for enzymatic determination of glucose or creatinine kinase activity when coupled with glucose-6-phosphate dehydrogenase.
Specification
|
Appearance |
White to light yellow amorphous powder, lyophilized |
|
Purity(SDS-PAGE) |
≥90.0% |
|
Enzyme powder specific activity |
≥45.0 U/mg |
|
NADH/NADPH oxidase |
≤0.01% |
|
ATPase |
≤0.03% |
|
Glucose-6-phosphate dehydrogenase |
≤0.01% |
Properties
|
EC number |
2.7.1.1 (Recombinant from microorganism) |
|
|
Molecular weight |
36 kDa (SDS-PAGE) |
|
|
Isoelectric point |
5.64 |
|
|
Michaelis constants |
3.2× 10-4 M (D-glucose) ，8.3× 10-5 M (ATP |
|
|
Inhibitors |
SDS, Ag+, Fe3+, Hg2+, Cu2+ |
|
|
Optimum pH |
8.0 |
Fig. 1 |
|
Optimum temperature |
45 ℃ |
Fig. 2 |
|
pH stability |
pH 6.5 – 9.0 (25 ℃, 16h) |
Fig. 3 |
|
Thermal stability |
Below 37 ℃ (pH 8.0, 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: 50 mM Tris-HCl, contains 13.3 mM MgCl2, pH 8.0.
Reagent II: 0.67 M glucose dissolved by Reagent I.
Reagent III: 16.5 mM ATP dissolved by Reagent I.
Reagent IV: 6.8 mM NAD dissolved by Reagent I.
Reagent V: 300 U/mL Glucose-6-phosphate dehydrogenase dissolved by Reagent I.
Enzyme diluent: 0. 1% BSA dissolved by Reagent I.
Sample: The enzyme was diluted to 0. 1-0.3 U/mL with enzyme diluent.
Procedure
1. Add 2.3 mL Reagent I, 0.5 mL Reagent II, 0. 1 mL Reagent III, 0. 1 mL Reagent IV, 0.01 mL Reagent V in 3mL cuvette (d= 1.0cm), preincubate the reaction mixture at 37℃ for 5min.
2. Add 0. 1mL the enzyme solution in the reaction mixture and mix.
3. Record the ΔAs at 340nm 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
Vt: Total volume (3. 11 mL)
Vs: enzyme volume (0. 1mL)
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. Colowick, S.P. (1973) The Enzymes (3rd Ed.), 4, 1–48.
2. Barnard, E.A. (1975) Methods Enzy mol., 42, 6–25.
3. Wright, C.L. and Warsy, A.S. (1978) Biochem. J., 175, 125– 135.
4. Li, S.J., Umena, Y., Matsuoka, T., Kita, A., Fukui, K. and Morimoto, Y. (2007) Biochem. Biophys. Res. Commun., 358, 1002– 1007.
