NIPRO ENZYMES
1GLUCOKINASE 2 (GlcK2)[EC 2. 7. 1. 2]from Recombinant E.coli
ATP + D-Glucose ↔ ADP + D-Glucose-6-phosphate
SPECIFICATIONStateSpecific activity
Contaminants

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Optimum temperature
Thermal stability
Michaelis constants
Substrate specificity: Lyophilized: more than 350 U/mg protein: (as GlcK2 activity = 100 %)Glucose-6-phosphate dehydrogenase
Phosphoglucomutase
6-Phosphogluconate dehydrogenase
Hexose-6-phosphate isomerase
Glutathione reductase: ca. 32,000: 9.0: 7.0 - 10.0
: 70 °C: No detectable decrease in activity up to 60 °C.
: (60 mM Tris-HCI buffer, pH 8.5, at 30 °C)
GlucoseATP: D-Glucose
D-Mannose
D-Fructose< 0.01 %< 0.01 %< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 5)
(Fig. 3, 4)
0.1 mM
0.05 mM
100 %20 %0%

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for diagnostic reagent, for example, glucose determination or CK determination, and forthe specific determination of glucose.

NIPRO ENZYMESASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
ATP + Glucose

GlcK2

ADP + Glucose-6-phosphate

Glucose-6-phosphate + NADP+G6PD

Gluconolactone-6-phosphate + NADPH + H+

Unit Definition
One unit of activity is defined as the amount of GlcK2 that forms 1 μmol of glucose-6-phosphate perminute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 9.0
Ⅱ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 NNaOH))
Ⅲ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O/100 mL distilled water)
Ⅳ NADP+ solution ; 22.5 mM [(0.172 g NADP+ monosodium salt or 0.177 g NADP+ disodium salt)/10mL distilled water]
Ⅴ Glucose solution ; 40 mM (0.072 g glucose (anhyd.)/10 mL distilled water)
Ⅵ Glucose-6-phosphate dehydrogenase (G6PDH) ; (from yeast. Roche Diagnostics K.K., No. 127671) suspension in 3.2 M (NH4)2SO4 solution (10 mg/2 mL) approx. 140 U/mg at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50 mM Tris-HCIbuffer, pH 8.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 17.97 mL
SolutionⅣ 1.20 mL
SolutionⅡ 1.20 mL
SolutionⅤ 9.00 mL
SolutionⅢ 0.60 mL
SolutionⅥ 0.03 mL
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.01 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (ΔAbs340) in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (3.00 + 0.01)
6.22 X 0.01

Specific activity (U/mg protein) =X d.f.

Volume activity (U/mL)
Protein concentration (mg/mL)*

d.f. ; dilution factor
6.22 ; millimolar extinction coefficient of NADPH (cm2/μmol)*Protein concentration; determined by Bradford's method
REFERENCE
1. Hengartner, H., and Zuber, H.; FEBS Lett., 37, 212 (1973)
2. Kamei, S., Tomita, K., Nagata, K., Okuno, H., Shiraishi, T., Motoyama, A., Ohkubo, A., andYamanaka, H.; J. Clin. Biochem. Nutr., 3,1 (1987)

NIPRO ENZYMES
3. Tomita, K., Kamei, S., Nagata, K., Okuno, H., Shiraishi, T., Motoyama, A., Ohkubo, A., andYamanaka, M.; ibid., 3, 11 (1987)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100

500456789101101256789101112pH

Fig. 1 pH profile

Fig. 2 pH stability△ acetate, ○ phosphate,● Tris-HCl, ▲ carbonate

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M), :△ acetate,○ phosphate,● Tris-HCl, ▲ carbonate

100

Remaining activity (%)

Remaining activity (%)

4pH10050050203040506070805000153045

Temperature (℃)

Time (min.)Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min. in 0.1 M
Tris-HCl buffer, pH 9.0

treated in 0.1 M Tris-HClbuffer , pH 9.0○ 60 ℃, □ 70 ℃, ● 80 ℃60

NIPRO ENZYMES

Relative activity (%)

10050020406080

Temperature (℃)
Fig. 5 Thermal activity
defined as 100 % at 70 ℃100