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Glucose-6-Phosphate Dehydrogenase - Assay

Source:
Leuconostoc mesenteroides
CAS:
9001-40-5
EC:
1.1.1.49
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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.

equation

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

Glucose-6-Phosphate Dehydrogenase Products

Description
Activity
Code
Cat. #
Size
Price
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
Code
ZFDP
LS004002
1 ku
$68.00
LS004004
10 ku
$515.00
LS004006
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 200 x 1 ku. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
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
Code
ZF
LS003983
500 un
$50.00
LS003985
5 ku
$325.00
LS003987
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 100000 x 1 ku. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
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
Code
ZFD
LS003992
900 un
$50.00
LS003993
9 ku
$325.00
LS003994
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 180000 x 1 ku. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
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
Code
ZFL
LS003981
1 ku
$90.00
LS003980
10 ku
$650.00
LS003982
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 200000 x 1 ku. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
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
Code
ZFLD
LS003997
2 ku
$90.00
LS003998
18 ku
$650.00
LS003999
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 360000 x 1 ku. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
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