Glucose-6-Phosphate Dehydrogenase Assay

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Glucose-6-Phosphate Dehydrogenase Assay

Method

This dehydrogenase is rather unique in that it possesses dual coenzyme specificity. When assayed under conditions that are optimal for the particular coenzyme, the ratio of observed catalytic activity is NAD/NADP = 1.8. The reaction velocity is determined by measuring the increase in absorbance at 340 nm resulting from the reduction of NAD or NADP. One unit reduces one micromole of pyridine nucleotide per minute at 30°C and pH 7.8 under the specified conditions.

Reagents

0.055 M Tris⋅HCl buffer pH 7.8 containing 0.0033 M magnesium chloride
0.006 M Nicotinamide adenine dinucleotide phosphate, monosodium salt, (NADP), (TPN). Note: NADP may vary in salt form and degree of hydration. Care must be exercised to use an analytical grade and the correct molecular weight. When using NAD, prepare 0.06 M solution.
0.1 M Glucose-6-phosphate. As above, an analytical grade and the correct molecular weight must be used.
5 mM Glycine buffer, pH 8.0
5 mM Glycine buffer, pH 8.0 containing 0.1% bovine serum albumin

Enzyme

Dissolve lyophilized enzyme or dilute enzyme suspension to 1.0 mg/ml in 5 mM glycine buffer, pH 8.0. Care should be taken not to shake the solution as precipitation may result. Dilute further immediately before use in 5 mM glycine buffer, containing 0.1% albumin, to obtain a rate of 0.02 - 0.045 ΔA/min.

Procedure

Adjust spectrophotometer to 340 and 30°C.

Pipette into each cuvette as follows:

0.055 M Tris⋅HCl buffer, pH 7.8 with 0.0033 M MgCl₂ 2.7 ml

0.006 M NADP (or 0.06 M NAD) 0.1 ml

0.1 M Glucose-6-phosphate 0.1 ml

Incubate in spectrophotometer at 30deg.C for 7 - 8 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml diluted enzyme and record increase in A₃₄₀/min for 4 to 5 minutes. Calculate �A₃₄₀/minute from the initial linear portion of the curve.

Calculation

\frac{\text{Units}}{\text{mg}}\text{ = }\frac{\frac{\Delta \text{A}_{340}}{\text{min}}}{\frac{\text{6.22 x mg enzyme}}{\text{ml reaction mixture}}}

Glucose-6-Phosphate Dehydrogenase Products

Description

Glucose-6-Phosphate Dehydrogenase, High-Activity, Suspension

Source:

Leuconostoc mesenteroides

Chromatographically purified for higher specific activity. Same as Code: ZF except assayed using NAD. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities 0.02%, 0.003%, 0.002% and 0.002% respectively. A suspension in 3.7M ammonium sulfate.

Store at 2-8°C.

…

Activity

≥600 NAD units per mg protein

Certificate of Analysis

Code

ZFDP

Product details

LS004002

1 ku

$68.00

LS004004

10 ku

$515.00

LS004006

Bulk

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Description

Glucose-6-Phosphate Dehydrogenase, Suspension

Source:

Leuconostoc mesenteroides

Chromatographically purified. A suspension in 3.7M ammonium sulfate. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities 0.02%, 0.003%, 0.002% and 0.002% respectively.

Store at 2-8°C.

…

Activity

≥200 NADP units per mg protein

Certificate of Analysis

Code

Product details

LS003983

500 un

$50.00

LS003985

5 ku

$325.00

LS003987

Bulk

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Description

Glucose-6-Phosphate Dehydrogenase, Suspension

Source:

Leuconostoc mesenteroides

Chromatographically purified. Same as Code: ZF except assayed using NAD. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities 0.011%, 0.002%, 0.0011% and 0.0011% respectively. A suspension in 3.7M ammonium sulfate.

Store at 2-8°C.

…

Activity

≥360 NAD units per mg protein

Certificate of Analysis

Code

ZFD

Product details

LS003992

900 un

$50.00

LS003993

9 ku

$325.00

LS003994

Bulk

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Description

Glucose-6-Phosphate Dehydrogenase, Lyophilized

Source:

Leuconostoc mesenteroides

Chromatographically purified. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities 0.02%, 0.003%, 0.002% and 0.002% respectively. A lyophilized powder.

Store at 2-8°C.

…

Activity

≥200 NADP units per mg protein

Certificate of Analysis

Code

ZFL

Product details

LS003981

1 ku

$90.00

LS003980

10 ku

$650.00

LS003982

Bulk

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Description

Glucose-6-Phosphate Dehydrogenase, Lyophilized

Source:

Leuconostoc mesenteroides

Chromatographically purified. Same as Code: ZFL except assayed using NAD. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities 0.011%, 0.002%, 0.0011% and 0.0011% respectively. A lyophilized powder.

Store at 2-8°C.

…

Activity

≥360 NAD units per mg protein

Certificate of Analysis

Code

ZFLD

Product details

LS003997

2 ku

$90.00

LS003998

18 ku

$650.00

LS003999

Bulk

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0.055 M Tris⋅HCl buffer, pH 7.8 with 0.0033 M MgCl₂	2.7 ml
0.006 M NADP (or 0.06 M NAD)	0.1 ml
0.1 M Glucose-6-phosphate	0.1 ml

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Glucose-6-Phosphate Dehydrogenase - Assay

Glucose-6-Phosphate Dehydrogenase Assay

Glucose-6-Phosphate Dehydrogenase Products