Phosphoglucomutase Assay

Method: Phosphoglucomutase catalyzes an intra-molecular phosphate transfer between the 1 and 6 positions of glucose and prepares phosphorylated compounds for subsequent energy-producing or biosynthetic reactions. Enzymatic activity is determined by measuring the increase in absorbance at 340 nm caused by the reduction of NAD in the following reactions:

One unit reduces 1 umole of NAD per minute at 30°C, pH 7.6, using glucose-1-phosphate as the substrate in a glucose-6-phosphate dehydrogenase coupled system.

Reagents

• 6.5 mg/ml solution NAD trihydrate (MW 717.5) in reagent grade water. Prepare fresh daily.
• 200 mg Glucose-1-phosphate dipotassium dihydrate (MW 372.4) in 5 ml reagent grade water
• 0.5 mg/ml Glucose-1,6-diphosphate, tetracyclohexylammonium salt (MW 808.9)
• Imadazole buffer: Dissolve 3.26 gms imidazole (MW 68.08) in 900 ml reagent grade water. Add 0.813 gms magnesium chloride and stir to dissolve. Add 0.645 gms EDTA disodium dihydrate and stir until completely dissolved. Adjust pH to 7.6 with 5N HCl. Bring to final volume of 1000 ml with reagent grade water.
• 150 u/ml Glucose-6-phosphate dehydrogenase (Worthington Code ZF) in imidazole buffer. Prepare fresh daily.

Enzyme

Dilute in buffer to approximately 0.2 u/ml, prepared immediately before use.

Procedure

Adjust the spectrophotometer to 340 nm and 30° C.

Mix together with stirring, no more than 2 hours before use, the following reaction mixture: 26.0 ml imidazole buffer, 1.0 ml NAD solution, 1.0 ml glucose-1-phosphate solution, 1.0 ml glucose-1,6-diphosphate solution, and 0.1 ml ZF. Place in a 30°C water bath to achieve temperature equilibration. Pipette into cuvettes 2.9 ml reaction mixture. Incubate in spectrophotometer at 30° C for 3-5 minutes to achieve temperature equilibrium and record blank rate, if any. Add 0.1 ml of appropriately diluted enzyme and record increase in absorbance at 340 nm for 5 - 7 minutes. Calculate ΔA₃₄₀ from the initial linear portion of the curve.

Calculation