PREPARATION and SPECIFICATION
| Appearance | White amorphous powder, lyophilized | |
|---|---|---|
| Activity | GradeⅢ 150 U/mg-solid or more | |
| Contaminants | Phosphoglucose isomerase | ≤1.0×10-1 % |
| 6-Phosphogluconate dehydrogenase | ≤1.0×10-2 % | |
| Glucose-6-phosphate dehydrogenase | ≤1.0×10-2 % | |
| Myokinase | ≤1.0×10-2 % | |
| Glutathione reductase | ≤5.0×10-1% | |
PROPERTIES
| Stability | Stable at −20 ℃ for at least one year(Fig.1) |
|---|---|
| Molecular weight | approx. 82,000 (by gel filtration) |
| Isoelectric point | 4.1±0.1 |
| Michaelis constants | 2.3×10-4 M (D-Glucose), 7.7×10-5 M (ATP) |
| Inhibitors | Metal ions, p-chloromercuribenzoate, iodoacetamide, SDS, etc |
| Optimum pH | 8.0−9.0(Fig.2) |
| Optimum temperature | 50 ℃(Fig.3) |
| pH Stability | pH 4.0−9.0 (25 ℃, 20 hr)(Fig.4) |
| Thermal stability | below 45 ℃ (pH 7.0, 30 min)(Fig.5) |
| Substrate specificity | (Table 1) |
| Effect of various chemicals | (Table 2) |
APPLICATIONS
The enzyme is useful for enzymatic determination of glucose, adenosine-5′-triphosphate (ATP) and creatine phosphokinase in combination with glucose-6-phosphate dehydrogenase (=G-6-PDH, G6D311, G6D-321).
ASSAY
Principle
The formation of NADH is measured at 340 nm by spectrophotometry.
Unit definition
One unit causes the formation of one micromole of NADH per minute under the conditions detailed below.
Method
Reagents
| A. Tris-HCl buffer, pH 8.0 | 50 mM, containing 13.3 mM MgCl2 | |
|---|---|---|
| B. Glucose solution | 0.67 M in Tris-HCl buffer solution (A) (The solution should be keep at room temperature for at least 1 hour before use) | |
| C. ATP solution | 16.5 mM in Tris-HCl buffer solution (A) (Should be freshly prepared) | |
| D. NAD＋ solution | 6.8 mM in Tris-HCl buffer solution (A) (Should be freshly prepared) | |
| E. G-6-PDH solution | 300 U/mL (Dilute with Tris-HCl buffer solution (A) and store on ice) | |
| F. Enzyme diluent | Tris-HCl 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 30℃ for approximately 5 minutes.
| 2.30 mL | Tris-HCl buffer solution | (A) |
| 0.50 mL | Glucose solution | (B) |
| 0.10 mL | ATP solution | (C) |
| 0.10 mL | NAD＋ solution | (D) |
| 0.01 mL | G-6-PDH solution | (E) |
| Concentration in assay mixture | |
|---|---|
| Tris-HCl buffer | 50 mM |
| Glucose | 0.11 M |
| ATP | 0.53 mM |
| NAD＋ | 0.22 mM |
| MgCl2 | 13 mM |
| BSA | 3.2 μg/mL |
| G-6-PDH | approx.1.0 U/mL |
2.Add 0.1ml 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 portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by the same method as the test except that the enzyme diluent (F) is added instead of the enzyme solution.
*Dissolve the enzyme preparation on ice-cold enzyme diluent (F) and dilute to 0.1−0.3 U/mL with the same buffer, immediately before the assay.
Calculation
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×5.0×df
Weight activity (U/mg) ＝ (U/ml)×1/C
| Vt | : Total volume (3.11 mL) |
| Vs | : Sample volume (0.1 mL) |
| 1.0 | : Light path length (cm) |
| 6.22 | : Millimolar extinction coefficient of NADH (cm2/micromole) |
| df | : Dilution factor |
| C | : Enzyme concentration in dissolution (c mg/mL) |
Table 1. Substrate specificity of Hexokinase
[Pyruvate kinase-Lactate dehydrogenase system with 0.1 M Tris-HCl buffer, pH 7.5]
Substrate(100mM) Relative activity(%) D-Glucose 100 D-Fructose 140 D-Mannose 52 2-Deoxy-D-glucose 91 Substrate(100mM) Relative activity(%) D-Galactose 0 D-Xylose 2 D-Glucosamine 58
Table 2. Effect of Various Chemicals on Hexokinase
[The enzyme dissolved in 50mM K-phosphate buffer, pH 6.5 (5 U/mL) contg. 0.1 % bovine serum albumin was incubated with each chemical at 30 ℃ for 1hr.]
Chemical Concn.(mM) Residual
activity(%)None – 100 Metal salt AgNO3 2.0 0 BaCl2 2.0 99 CaCl2 2.0 98 CdCl2 2.0 85 CoCl2 2.0 85 CuSO4 2.0 25 FeCl3 2.0 28 FeSO4 2.0 80 HgCl2 2.0 0 MgCl2 2.0 98 MnCl2 2.0 100 NiCl2 2.0 100 Pb(OAc)2 2.0 98 Zn(OAc)2 2.0 98 ZnSO4 2.0 99 NaF 20.0 101 NaN3 20.0 102 Chemical Concn.(mM) Residual
activity(%)PCMB 2.0 0 MIA 2.0 80 IAA 2.0 7 EDTA 5.0 103 (NH4)2SO4 20.0 104 Borate 20.0 102 o-Phenanthroline 2.0 101 α,α′-Dipyridyl 2.0 102 Urea 2.0 104 Guanidine 2.0 103 Hydroxylamine 2.0 104 Na-cholate 1.0 % 102 Triton X-100 1.0 % 105 Brij 35 1.0 % 0 SDS 0.1 % 25 Tween 20 0.1 % 101 Span 20 0.1 % 106 DAC 0.1 % 101
Ac, CH3CO; NEM, N-Ethylmaleimide; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.
Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. pH-Activity
30 ℃ in the 50 mM buffer solution: pH 6.2-7.5, PIPES-NaOH: pH 7.5-9.0, Tris-HCI: pH 9.0-10.0, Glycine-NaOH
Fig.3. Temperature activity
(in 50 mM Tris-HCI buffer,pH 8.0)
Fig.4. pH-Stability
25 ℃, 20 hr-treatment in the 0.1 M buffer solution: pH 4.0-8.0,Acetate-NaOH;pH 6.0-8.0, K-phosphate; pH 7.5-9.0,Tris-HCl;pH 9.0-10.5, Glycine-NaOH enzyme concn.: ca.10 U/mL
Fig.5. Thermal stability
30 min-treatment with 50 mM K-phosphate buffer, pH 7.0, containing 0.1% bovine serum albumin enzyme concn.: ca.5 U/mL
| Size | 1 MG, 10 MG, 5 MG |
|---|
Related Products
| Name | Galactokinase; BiGalK |
| Product Code | EN01018 |
| E.C. | 2.7.1.6 |
| Product Description | E. coli recombinant galactokinase from Bifidobacterium infantis |
| Unit Definition | One unit is defined as the amount of enzyme that catalyzes the formation of 1 μmol of Gal-1-P from galactose and ATP per minute at 37 °C. |
10 in stock
| Name | UDP-Glc dehydrogenase; UDPDH |
| Product Code | EN01020 |
| E.C. | 1.1.1.22 |
| Product Description | E. coli recombinant uridine-5-diphosphoglucose dehydrogenase from Streptococcus pyogenes |
| Unit Definition | One unit will oxidize 1.0 μmole of UDP-glucose to UDP-glucuronic acid per minute at pH 8.7 at 25 °C. |
10 in stock
| Name | α2,6-sialytransferase（Pd26ST） |
| Catalog Number | EN01001 |
| E.C. | 2.4.99.1 |
| Product Description | E.coli recombinant α2,6-sialytransferase from Photobacterium damsel. |
| Unit Definition | One unit is define as the amount of enzyme that catalyzes the formation of 1 μmol Siaα2,6Lac from CMP-Sia and lactose per minute at 37℃. |
10 in stock
| Name | N-acetylglucosamine-1-Puridyltransferase; AGX1 |
| Product Code | EN01021 |
| E.C. | 2.3.1.157 |
| Product Description | E. coli recombinant N-acetylglucosamine-1-Puridyltransferase from Homo sapien |
| Unit Definition | One unit is defined as the amount of enzyme that catalyzes the formation of 1 μmol of UDP-GlcNAc from GlcNAc-1-P and UTP per minute at 37 °C. |
10 in stock
Reviews
There are no reviews yet.