CODE No.:M1P211

6-PHOSPHOGLUCONATE DEHYDROGENASE
(DECARBOXYLATING)
(6PGDH)

[EC 1.1.1.44]
from Microorganism

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 40 U/mg protein
Contaminants	:	(as 6PGDH activity = 100%)
		Glucokinase	<0.01%
		Phosphoglucomutase	<0.01%
		Hexose-6-phosphate isomerase	<0.01%
		Glutathione reductase	<0.01%

PROPERTIES

Molecular weight	:	ca. 132,000
Subunit molecular weight	:	ca. 33,000
Optimum pH	:	7.0 - 7.5	(Fig.1)
pH stability	:	5.0 - 10.0	(Fig.2)
Isoelectric point	:	ca. 4.5
Thermal stability	:	(50mM MES-NaOH buffer, pH 6.8, containing 0.5 M KCl)
		No detectable decrease in activity up to 40°C.	(Fig.3, 4)
Michaelis constants	:	(80mM GIycylglycine buffer, pH 7.5, at 30°C)
		6-Phospho-D-gluconate	0.95mM
		NAD+	0.32mM
Stabilizer	:	KCl, MgCl2, Sorbitol, BSA
Activators	:	Mg2+, Mn2+, Ca2+, K+, Na+
Inhibitors	:	Fructose 1,6-bisphosphate, Erythrose 4-phosphate, NADH

STORAGE

stable at -20°C for at least six months

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 6PGDH that forms 1 µmol of NADH per minute at 30°C.

Solutions

1. Buffer solution ; 100mM Glycylgycine-NaOH, pH 7.5
2. 6-Phospho-D-gluconate (6PG) solution ; 100mM (0.378g 6PG trisodium salt·2H₂O/10mL distilled water)
3. NAD⁺ solution ; 50mM (0.332 g NAD⁺ free acid/10mL distilled water)
4. MgCl₂ solution ; 1 M (20.33 g MgCl₂·6H₂O/100mL distilled water)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 100mM MES-NaOH buffer containing 1 mg/mL BSA, pH 6.8.

Procedure

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

   Solution I	24.6mL	Solution III	2.1mL
   Solution II	3.0mL	Solution IV	0.3mL

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

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