Enzymatic Assay of Hexokinase

1. Objective

To standardize a procedure for the enzymatic determination of Hexokinase.

2. Scope

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

3. Definitions


4. Discussion

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

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. Safety

Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

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

Continuous Spectrophotometric Rate Determination


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

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

C    19 mM Adenosine 5'-Triphosphate Solution (ATP)
            (Prepare 10 mL in deionized water using adenosine 5' triphosphate, disodium Salt, Sigma Product No. A2383. PREPARE FRESH.)

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

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, Sigma Product No. N0505. PREPARE FRESH.)

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, Sigma Product No. G4134, in cold Reagent A.)3

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:

  Test Blank3
Reagent A (Buffer) 1.00 1.00
Reagent B (D-Glucose) 1.00 1.00
Reagent C (ATP) 0.10 0.10
Reagent D (MgCl2) 0.20 0.20
Reagent E (β-NADP) 0.20 0.20
Reagent F (G-6-PDH) 0.02 0.02


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

Deionized Water ------ 0.05
Reagent G (Enzyme Solution) 0.05 ------


    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.


  Units/mL enzyme = (ΔA340nm/min Test - ΔA340nm/min Blank)(2.57)(df)


    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

  Units/mg solid = Units/mL enzyme
mg solid/mL enzyme


  Units/mg protein = Units/mL enzyme
mg protein/mL enzyme


One unit will phosphorylate 1.0 μmole 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.

8. References

Bergmeyer, H.U., Grassl, M., and Walter, H.E. (1983) in Methods of Enzymatic Analysis (Bergmeyer, H.U. ed) 3rd ed., Volume II, 222-223, Verlag Chemie, Deerfield Beach, FL


  1. This procedure is not to be used to assay the activity of Hexokinase, Sigma Product No. H3779, Hexokinase, Insoluble enzyme attached to beaded agarose, Sigma Product No. H2005, and Hexokinase, Insoluble enzyme attached to polyacrylamide, Sigma Product No. H8254.
  2. Glucose-6-Phosphate Dehydrogenase unit definition: One unit will oxidize 1.0 μmole 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.


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