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.
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.
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