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Hexokinase Assay

Method: The assay is based upon the reduction of NAD+ through a coupled reaction with glucose-6phosphate 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 30C 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 TrisMgCl2 buffer 6.8 mM NAD in above TrisMgCl2 buffer 16.5 mM Adenosine 5'Triphosphate in above TrisMgCl2 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 TrisMgCl2 buffer. Store at 0 - 4C during use. Enzyme Dissolve in TrisMgCl2 buffer, pH 8.0 to obtain a rate of 0.02 - 0.04 A/min. Procedure Adjust spectrophotometer to 340 nm and 30C. Pipette into each cuvette as follows: TrisMgCl2 buffer 0.67 M Glucose 16.5 mM ATP 6.8 mM NAD 2.28 ml 0.50 ml 0.10 ml 0.10 ml

G-6-PDH 0.01 ml Incubate in the spectrophotometer at 30C 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 A/min from initial linear portion of curve.

In the first reaction of glycolysis, the gama-phosphoryl group of an ATP molecule is transferred to the oxygen at the C-6 of glucose (magnesium ion is required as the reactive form of ATP is the chelated complex with magnesium (II) ion). This step is a direct nucleophilic attack of the hydroxyl group on the terminal phosphoryl group of the ATP molecule (Aleshin, 99). This produces glucose-6-phosphate and ADP. Hexokinase is the enzyme that catalyzes this phosphoryl-group-transfer. Hexokinase undergoes and induced-fit conformational change when it binds to glucose, which ultimately prevents the hydrolysis of ATP. It is also allosterically inhibited by physiological concentrations of its immediate product, glucose-6-phosphate. This is a mechanism by which the influx of substrate into the glycolytic pathway is controlled.