Hexokinase Assay

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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.

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

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

Hexokinase Products

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

Certificate of Analysis

Code

HKQLR

Product details

LS002515

2.5 ku

$66.00

LS002516

10 ku

$220.00

LS002518

Bulk

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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

Certificate of Analysis

Code

HKQL

Product details

Minimum order quantity

200000

LS002514

Bulk

---

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

Certificate of Analysis

Code

HKQS

Product details

Minimum order quantity

200000

LS002500

Bulk