CODE No.:B5P111

PHOSPHOGLUCOSE ISOMERASE (PGI)

[EC 5.3.1.9]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 400 U/mg protein
Contaminants	:	(as PGI activity = 100%)
		Phosphofructokinase	<0.01%
		6-Phosphogluconate dehydrogenase	<0.01%
		Phosphoglucomutase	<0.01%
		NADPH oxidase	<0.01%
		Glutathione reductase	<0.01%

PROPERTIES

Molecular weight	:	ca. 200,000
Subunit molecular weight	:	ca. 54,000
Optimum pH	:	9.0 - 10.0	(Fig.1)
pH stability	:	6.0 - 10.5	(Fig.2)
Isoelectric point	:	4.2
Thermal stability	:	No detectable decrease in activity up to 60°C.	(Fig.3, 4)
Michaelis constants	:	(95mM Tris-HCl buffer, pH 9.0, at 30°C)
		Fructose 6-phospate	0.27mM

STORAGE

stable at -20°C for at least one year

ASSAY

Principle

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

Unit Definition

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

Solutions

1. Buffer solution ; 100mM Tris-HCl, pH 9.0
2. Fructose 6-phosphate (F6P) solution ; 100mM (0.310 g F6P disodium salt/10mL distilled water)
3. NADP⁺solution ; 22.5 mM (0.188 g NADP⁺sodium salt·4H₂O/10mL distilled water)
4. Glucose-6-phosphate dehydrogenase (G6PDH) ; (from yeast, Roche Diagnostics K.K., No. 127 671) 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 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	28.44mL	Solution III	0.60mL
   Solution II	0.90mL	Solution IV	0.06mL

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 the curve.

Calculation

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

REFERENCE

1. Muramatsu, N., and Nosoh, T.; Arch. Biochem. Biophys., 144, 245 (1971)

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