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HomeEnzyme Activity AssaysEnzymatic Assay of Phosphoglucomutase (EC 5.4.2.2)

Enzymatic Assay of Phosphoglucomutase (EC 5.4.2.2)

Description

This procedure may be used for all Phosphoglucomutase products except for β‑Phosphoglucomutase, Catalog Number P4109.

The continuous spectrophotometric rate detemination (A340, Light path = 1 cm) is based on the following reactions:

Where:

β-NADP – β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form

β-NADPH – β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form

G-6-PDH – Glucose-6-Phosphate Dehydrogenase

Unit Definition – One unit of phosphoglucomutase will convert 1.0 µmole of α-D-Glucose-1‑Phosphate to α-D-Glucose-6‑Phosphate per minute at pH 7.4 at 30 °C.

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Gly-Gly (Catalog Number G1002)
α-D-Glucose 1-Phosphate, dipotassium salt, hydrate (Catalog Number G6875)
β-Nicotinamide Adenine Dinucleotide Phosphate, sodium salt, hydrate (Catalog Number N0505)
α-D-Glucose 1,6-bisphosphate, tetra(cyclohexylammonium) salt, hydrate (Catalog Number G5875)
1.00 M Magnesium Chloride Solution (Catalog Number M1028)
L-Cysteine hydrochloride, monohydrate (Catalog Number C7880)
Sodium bicarbonate (Catalog Number S8875)
Glucose-6-Phosphate Dehydrogenase from baker’s yeast (Catalog Number G6378 or G4134)
Cuvettes and thermostatted spectrophotometer

Preparation Instructions
(Storage/Stability)

Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.

Buffer (0.25 M Glycylglycine Buffer, pH 7.4 at 30 °C) – Prepare a 33 mg/ml solution in ultrapure water using Gly-Gly (Catalog Number G1002). Adjust the pH of the solution to 7.4 at 30 °C using 5 M or 1 M HCl.

G-1-P Solution (0.15 M Glucose-1-Phosphate) – Prepare a 59.9 mg/ml solution in purified ultrapure using α-D-Glucose-1‑Phosphate, dipotassium salt, hydrate (Catalog Number G6875).
Note: The concentration was calculated using molecular weight corrected based upon maximum % water, % potassium, % glucose-1,6-diphosphate, and % purity.

β-NADP Solution (20 mM β-Nicotinamide Adenine Dinucleotide Phosphate) – Prepare a 18.9 mg/ml solution in ultrapure water using β-Nicotinamide Adenine Dinucleotide Phosphate, sodium salt, hydrate (Catalog Number N0505).
Note: The concentration was calculated using molecular weight corrected based upon maximum % water, % solvent, % sodium, and % purity.

G-1,6-P Solution (23.2 mM Glucose-1,6-bisphosphate) – Prepare a 22 mg/ml solution in ultrapure water using α-D-Glucose 1,6-bisphosphate, tetra(cyclohexylammonium) salt, hydrate (Catalog Number G5875).
Note: The concentration was calculated using molecular weight corrected based upon maximum % water, % solvent, % cyclohexylamine salt, and % purity.

MgCl2 Solution (0.9 M Magnesium Chloride) – Prepare a 1:1.11 dilution using 1.0 M Magnesium chloride solution (Catalog Number M1028) in ultrapure water.

Cysteine Solution (0.26 M L-Cysteine Hydrochloride) – Prepare a 53.8 mg/ml solution in ultrapure water using L-Cysteine hydrochloride, monohydrate (Catalog Number C7880). Adjust the pH of the solution to 7.0 using solid Sodium bicarbonate (Catalog Number S8875).
Note: The concentration was calculated using molecular weight corrected based upon maximum % water, % hydrochloride, and % purity.

G-6-PDH Solution (100 units/ml, Glucose-6-Phosphate Dehydrogenase) – Immediately before use, prepare a solution containing ~100 units/ml in cold Buffer using Glucose-6‑Phosphate Dehydrogenase (Catalog Number G6378 or G4134).

Enzyme Solution (Phosphglucomutase) – Immediately before use, prepare a solution containing 0.10–0.35 unit/ml of Phosphoglucomutase in cold Buffer.

Procedure

Final Assay Concentrations – In a 3.00 ml reaction mix, the final concentrations are 0.17 M Glycylglycine, 5.0 mM Glucose-1‑Phosphate, 0.67 mM β‑Nicotinamide Adenine Dinucleotide Phosphate, 0.39 mM Glucose-1,6‑bisphosphate, 30 mM Magnesium Chloride, 43 mM L-Cysteine, 1 unit of Glucose-6-Phosphate Dehydrogenase, and 0.005‑0.035 unit of Phosphoglucomutase.

1. Prepare a Reaction Cocktail by pipetting the following reagents into a suitable container:

2. Mix and equilibrate the Reaction Cocktail to 30 °C in a suitably thermostatted water bath. Adjust the pH to 7.4 at 30 °C using 1 M NaOH or 1 M HCl.

3. Pipette the following reagents into suitable cuvettes:

4. Mix by inversion and equilibrate to 30 °C in a suitably thermostatted spectrophotometer blanked versus air. Then add:

5. Immediately mix by inversion and record the increase in A340 for ~10 minutes using a suitably thermostatted spectrophotometer. Obtain the ΔA340/minute for all Tests and the Blank using the maximum linear rate over a one minute interval using a minimum of 4 points.

Results

Calculations

1.

Where:

3.00 = Total volume (ml) of assay

df = Dilution Factor

6.22 = Millimolar extinction coefficient of β-NADPH at 340 nm

0.10 = Volume (ml) of Enzyme Solution (Phosphoglucomutase) used in step 4

2.

Materials
Loading
1.
Bergmeyer HU. (New York, NY: 1974). et al., Methods of Enzymatic Analysis, Bergmeyer, H.U., ed., Volume 1, 2nd ed., Academic Press, Inc., 499-500..
2.
LOWRY W. 1969. GAMMA-GLOBULIN FOR BURKITT'S LYMPHOMA?. The Lancet. 294(7626):910. http://dx.doi.org/10.1016/s0140-6736(69)92373-3