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