CODE No.:B1G111

GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PDH)

[EC 1.1.1.49]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 150U/mg protein
Contaminants	:	(as G6PDH activity =100%)
		Glucokinase	<0.02%
		Phosphoglucomutase	<0.01%
		6-Phosphogluconate dehydrogenase	<0.02%
		Hexose-6-phosphate isomerase	<0.01%
		Glutathione reductase	<0.01%

PROPERTIES

Molecular weight	:	ca. 195,000
Subunit molecular weight	:	ca. 53,000
Optimum pH	:	8.7	(Fig.1)
pH stability	:	7.5 - 11.0	(Fig.2)
Isoelectric point	:	6.5 - 6.8
Optimum temperature	:	70°C
Thermal stability	:	No detectable decrease in activity up to 60°C.	(Fig.3, 4)
Michaelis constants	:	(84 mM Tris-HCl buffer, pH 9.0, at 30°C)
		Glucose 6-phosphate	0.16mM
		NADP+	0.016mM
		NAD+	1.64mM
Substrate specificity	:	Glucose 6-phosphate	100%
		2-Deoxyglucose 6-phosphate	0%
		Mannose 6-phosphate	0%
		Fructose 6-phosphate	0%
		Glucose 1-phosphate	0%
		Glucosamine 6-phosphate	0%
		6-Phosphogluconate	0%
	:	NADP+	100%
		NAD+	115%
Effecters	:	cations and anions	(Fig. 5,6)

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.

ASSAY

Principle

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

Unit Definition

One unit of activity is defined as the amount of G6PDH that forms 1 μmol of NADPH per minute at 30°C.

Solutions

1. Buffer solution ; 100mM Tris-HCl, pH 9.0
2. MgCl₂ solution ; 1 M (20.33 g MgCl₂·6H₂O/100mL distilled water)
3. NADP⁺ solution ; 22.5 mM (0.188g NADP⁺ sodium salt·4H₂O/10mL distilled water)
4. Glucose 6-phosphate (G6P) solution ; 33 mM (0.112g G6P disodium salt·2H₂O/10mL distilled water)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50mM Tris-HCl buffer, pH 8.5.

Procedure

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

   Solution I	25.20mL	Solution III	1.20mL
   Solution II	1.20mL	Solution IV	2.40mL

2. Incubate at 30°C for about 3minutes.
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 NADPH (cm²/μmol)
• *Protein concentration ; determined by Bradford's method

REFERENCE

1. Okuno. H., Nagata, K., and Nakajima, H.; J. Appl Biochem., 7.192 (1985)

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