To standardize a procedure for the enzymatic determination of hexokinase.


This procedure applies to most products1 that have a specification for Hexokinase determination by enzymatic determination.


ATP = Adenosine 5'-Triphosphate
ADP = Adenosine 5'-Diphosphate
G-6-PDH = Glucose-6-Phosphate Dehydrogenase
β-NADP = β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form
β-NADPH = β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form
6-PG = 6-Phospho-D-Gluconate


D-Glucose + ATP Hexokinase > D-Glucose 6-Phosphate + ADP
D-Glucose 6-Phosphate + β-NADP G-6-PDH >6-PG + β-NADPH


CONDITIONS: T = 25 °C, pH = 7.6, A340nm, Light path = 1 cm

METHOD: Continuous Spectrophotometric Rate Determination


Reagent A: 50 mM triethanolamine buffer, pH 7.6 at 25 °C (Buffer). Prepare 100 mL in deionized water using Triethanolamine Hydrochloride, Product No. T1502. Adjust to pH 7.6 at 25 °C with 1 M NaOH.

Reagent B: 555 mM D-Glucose Solution (D-Glucose). Prepare 10 mL in Reagent A using D-(+)-glucose, anhydrous (Product No. G8270.)

Reagent C: 19 mM adenosine 5'-triphosphate solution (ATP). Prepare 10 mL in deionized water using adenosine 5' triphosphate, disodium Salt (Product No. A2383, PREPARE FRESH).

Reagent D: 100 mM Magnesium Chloride Solution (MgCl2). Prepare 5 mL in deionized water using magnesium chloride solution (Product No. M1028.)

Reagent E: 14 mM β-nicotinamide adenine dinucleotide phosphate, oxidized form, solution (β-NADP). Prepare 10 mL in deionized water using β-Nicotinamide Adenine Dinucleotide Phosphate, Sodium Salt (Product No. N0505, PREPARE FRESH).

Reagent F: Glucose-6-Phosphate Dehydrogenase Enzyme Solution (G 6 PDH).2 Immediately before use, prepare a solution containing approximately 125 units/mL of glucose-6-phosphate dehydrogenase, Product No. G4134, in cold Reagent A.3

Reagent G: Hexokinase Enzyme Solution. Immediately before use, prepare a solution containing 0.5 - 1.0 unit/mL of hexokinase in cold deionized water.)


Pipette (in milliliters) the following reagents into suitable cuvettes:

Mix by inversion and equilibrate to 25 °C. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Immediately mix by inversion and record the increase in A340nm for approximately 5 minutes. Obtain the ΔA340nm/minute using the maximum linear rate for both the Test and Blank.


2.57 = Total volume (in milliliters) of assay
df = Dilution factor
6.22 = Millimolar extinction coefficient of β-NADPH at 340 nm
0.05 = Volume (in milliliter) of enzyme used

One unit will phosphorylate 1.0 μmol of D-glucose per minute at pH 7.6 at 25 °C.

In a 2.57 mL reaction mix, the final concentrations are 39 mM triethanolamine, 216 mM D-glucose, 0.74 mM adenosine 5'-triphosphate, 7.8 mM magnesium chloride, 1.1 mM β-nicotinamide adenine dinucleotide phosphate, 2.5 units glucose 6 phosphate dehydrogenase, and 0.025 - 0.05 unit of hexokinase.


  1. This procedure is not to be used to assay the activity of Hexokinase, Product No. H3779, Hexokinase, Insoluble enzyme attached to beaded agarose, Product No. H2005, and Hexokinase, Insoluble enzyme attached to polyacrylamide, Product No. H8254.
  2. Glucose-6-phosphate dehydrogenase unit definition: One unit will oxidize 1.0 μmol of D-glucose 6-phosphate to 6-phospho-D-gluconate per minute in the presence of β-NADP at pH 7.4 at 25 °C.
  3. Other types of glucose-6-phosphate dehydrogenase may contain varying amounts of hexokinase as an impurity. This may cause a high blank rate , therefore, it is imperative that the G-6-PDH has low levels of hexokinase impurity.


Bergmeyer H, Grassl M, Walter H. 1983. in Methods of Enzymatic Analysis. (Bergmeyer, H.U. ed). 3(II):222-223.