Enzymatic Assay of Hexokinase

Objective

To standardize a procedure for the enzymatic determination of hexokinase.

Scope

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

Definitions

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

reaction

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

Procedure

CONDITIONS: T = 25 °C, pH = 7.6, A_340nm, Light path = 1 cm

METHOD: Continuous Spectrophotometric Rate Determination

REAGENTS:

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.)

TEST METHOD:

    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 A_340nm 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 A_340nm for approximately 5 minutes. Obtain the ΔA_340nm/minute using the maximum linear rate for both the Test and Blank.

CALCULATIONS:

	Units/mL enzyme =	(ΔA340nm/min Test - ΔA340nm/min Blank)(2.57)(df)
		(6.22)(0.05)

   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

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

FINAL ASSAY CONCENTRATION :
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.

Notes

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.

Reference

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