CODE No.:Z2G111

GLUCOKINASE (ZM-GlcK)

[EC 2.7.1.2]
from Zymomonas mobilis

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 150 U/mg protein
Contaminants	:	(as ZM-GlcK activity = 100%)
		Glucose-6-phosphate dehydrogenase	<0.02%
		Phosphoglucomutase	<0.01%
		6-Phosphogluconate dehydrogenase	<0.01%
		Hexose-6-phosphate isomerase	<0.01%
		Glutathione reductase	<0.01%

PROPERTIES

Molecular weight	:	ca. 66,000
Subunit molecular weight	:	ca. 33,000
Optimum pH	:	7.0 - 8.0	(Fig.1)
pH stability	:	6.0 - 8.0	(Fig.2)
Thermal stability	:	No detectable decrease in activity up to 40 °C.	(Fig.3, 4)
Michaelis constants	:	(60mM Phosphate buffer, pH 7.0, at 30°C)
		Glucose	0.10mM
		ATP	0.65mM
Activator	:	Pi

STORAGE

stable at -20 °C for at least one year

APPLICATION

The enzyme is useful for diagnostic reagent, for example, glucose determination or CK determination, and for the specific determination of glucose.
Tris-HCl buffer is not suitable for the practical use of ZM-GlcK.

ASSAY

Principle

The change in absorbance is measured at 340 nm according to the following reactions.

Unit Definition

One unit of activity is defined as the amount of ZM-GlcK that forms 1 μmol of glucose 6-phosphate per minute at 30°C.

Solutions

1. Buffer solution ; 100mM Triethanolamine - NaOH and 3 mM K₂HPO₄, pH 7.5
2. ATP solution ; 100mM (0.605 g ATP disodium salt·3H₂O/(8.2 mL distilled water + 1.8 mL 1 N-NaOH))
3. MgCl₂ solution ; 1 M (20.33 g MgCl₂·6H₂O/100mL distilled water)
4. NAD⁺ solution ; 100mM (0.663 g NAD⁺ free acid/10mL distilled water)
5. Glucose solution ; 40mM (0.072 g glucose (anhyd.)/10mL distilled water)
6. Glucose-6-phosphate dehydrogenase (G6PDH) ; 2000 U/mL (from Zymomonas mobilis, Unitika Ltd., Dissolve with Buffer solution I)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50mM potassium phosphate buffer containing 1 mg/mL BSA, pH 7.0.

Procedure

1. Prepare the following reaction mixture and pipette 3.00mL of reaction mixture into a cuvette.

   Solution I	20.07mL	Solution IV	0.60mL
   Solution II	1.50mL	Solution V	7.50mL
   Solution III	0.30mL	Solution VI	0.03mL

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

• d.f. ; dilution factor
• 6.22 ; millimolar extinction coefficient of NADH (cm²/μmol)
• *Protein concentration ; determined by Bradford's method

REFERENCE

1. Scopes. R.K., Testolin, V., Stoter, A., Griffiths-Smith, K., and Algar, E.M.; Biochem. J., 228, 627 (1985)

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