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:

Reagent Volume (ml)
Buffer 40.8
G-1-P Solution 2.0
β-NADP Solution 2.0
G-1,6-P Solution 1.0
MgCl2 Solution 2.0
Cysteine Solution 10.0
Total Volume 57.8

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:

Reagent Volume (ml)
  Blank Test 1 Test 2 Test 3
Reaction Cocktail 2.89 2.89 2.89 2.89
G-6-PDH Solution 0.01 0.01 0.01 0.01

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

Reagent Volume (ml)
  Blank Test 1 Test 2 Test 3
Buffer 0.10 0.05 0.03
Enzyme Solution 0.05 0.07 0.10

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

     

References

  1. Bergmeyer, H.U. et al., Methods of Enzymatic Analysis, Bergmeyer, H.U., ed., Volume 1, 2nd ed., Academic Press, Inc., (New York, NY: 1974) 499-500.
  2. Lowry, O.H., and Passonneau., J.V., Journal of Biological Chemistry, 244, 910-916 (1969).

 

10/17-1

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