Hexokinase - Assay

Source:
Yeast
CAS:
9001-51-8
EC:
2.7.1.1

Hexokinase Assay

Method
The assay is based upon the reduction of NAD+ through a coupled reaction with glucose-6-phosphate dehydrogenase and is determined spectrophotometrically by measuring the increase in absorbance at 340 nm.

formula

One unit of activity reduces one micromole of NAD+ per minute at 30°C and pH 8.0 under the specified conditions.

Reagents
  • 0.05 M Tris*HCl buffer, pH 8.0 with 13.3 mM MgCl2
  • 0.67 M Glucose in above Tris⋅MgCl2 buffer
  • 16.5 mM Adenosine 5'Triphosphate in above Tris⋅MgCl2 buffer
  • 6.8 mM NAD in above Tris⋅MgCl2 buffer

Note: NAD may vary in salt form and degree of hydration. Care should be exercised to use an analytical grade and the correct molecular weight.

Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (Worthington Code: ZF or ZFL). Dissolve at a concentration of 300 IU/ml in above Tris⋅MgCl2 buffer. Store at 0 - 4°C during use.

Enzyme

Dissolve in Tris⋅MgCl2 buffer, pH 8.0 to obtain a rate of 0.02 - 0.04 ΔA/min.

Procedure

Adjust spectrophotometer to 340 nm and 30°C.

Pipette into each cuvette as follows:

Tris⋅MgCl2 buffer 2.28 ml
0.67 M Glucose 0.50 ml
16.5 mM ATP 0.10 ml
6.8 mM NAD 0.10 ml
G-6-PDH 0.01 ml

Incubate in the spectrophotometer at 30°C for 6 - 8 minutes to achieve temperature equilibration and establish blank rate, if any. At zero time, add 0.1 ml of diluted hexokinase solution and mix thoroughly. Record increase in A340 for 3-4 minutes. Determine ΔA/min from initial linear portion of curve.

Calculation

Hexokinase Products

Description
Activity
Code
Cat. #
Size
Price
Description
Hexokinase, Lyophilized, Recomb
Source:
Recombinant Hexokinase from Yeast
A lyophilized powder. Phosphohexose isomerase, 6-phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities are 0.10%, 0.005%, 0.005% and 0.005%, respectively.
Store at -20°C.
Activity
≥150 units per mg protein
Code
HKQLR
LS002515
2.5 ku
$66.00
LS002516
10 ku
$220.00
LS002518
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
Hexokinase, Lyophilized
Source:
Yeast
Chromatographically purified. A lyophilized powder containing ~30% buffer/stabilizer. Phosphohexose isomerase, 6-phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities are 0.10%, 0.005%, 0.005% and 0.005%, respectively.
Store at -20°C.
Activity
≥150 units per mg protein
Code
HKQL
Minimum order quantity
200000
LS002514
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
Hexokinase, Suspension
Source:
Yeast
Chromatographically purified. A suspension in 2.8M ammonium sulfate. Phosphohexose isomerase, 6-phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities are 0.05%, 0.003%, 0.005% and 0.005%, respectively.
Store at 2-8°C.
Activity
≥150 units per mg protein
Code
HKQS
Minimum order quantity
200000
LS002500
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.