The enzyme is useful for the determination of α-amylase and inorganic phosphate in clinical analysis.
PROPERTIES
Molecular weight: ca. 34 kDa (gel filtration)
Structure: monomer of ca. 25 kDa (SDS-PAGE)
Michaelis constant: 2.3×10^−4 M (β-d-glucose-1-phosphate)
pH Optimum: ca. 7.0 (Fig. 1)
pH Stability: 5.0–9.5 (Fig. 2)
Optimum temperature: 40°C (Fig. 3)
Thermal stability: below 45°C (Fig. 4)
Stability (liquid form): stable at 37°C for at least one week (Fig. 5)
Stability (powder form): stable at 30°C for at lest one month (Fig. 6)
Activators: Mg2+, Mn2+, Co2+, Ni2+
Inhibitors: Hg2+, Zn2+, Cu2+, Cd2+
STABILIZERS: lactose, EDTA
ASSAY PROCEDURE
Principle
The appearance of NADPH is measured spectrophotometrically at 340 nm.
Reagents
A. HEPES–NaOH buffer, 0.3 M; pH 7.0, containing 40 mM KCl, 4 mM MgCl2 and 1.6% (w/v) Triton X-100: dissolve 7.15 g of HEPES, 298 mg of KCl, 81.3 mg of MgCl2·6H2O and 1.6 g of Triton X-100 in 75 ml of distilled water, adjust to pH 7.0 with 4 N NaOH and dilute with distilled water to 100 ml.
B. d-Glucose-1,6-bisphosphate (G-1,6-P2) solution, 3.0 mM : 60.7 mg of G-1,6-P2 cyclohexylammonium·4H2O/ 25 ml of distilled water.
C. NADP+ solution, 12 mM: 230 mg of NADP+·Na/25 ml of distilled water.
D. β-d-Glucose-1-phosphate (β-G-1-P) solution, 22 mM:167 mg of β-G-1-P disodium salt/25 ml of distilled water.
E. Glucose-6-phosphate dehydrogenase (G6PDH) solution: 1750 U/ml.
F. Enzyme dilution buffer: mix 10 mM KH2PO4 solution and 10 mM K2HPO4 solution to make a pH 7.0 solution.
Sample: dissolve the lyophilized enzyme to a volume activity of 1.0–3.0 U/ml with ice-cold enzyme dilution buffer (Reagent F) immediately before measurement.
Procedure
1. Pipette the following reagents into a cuvette (light path: 1 cm).
1.5 ml HEPES–NaOH buffer (Reagent A)
0.3 ml G-1,6-P2 solution (Reagent B)
0.3 ml NADP+ solution (Reagent C)
0.3 ml β-G-1-P solution (Reagent D)
0.02 ml G6PDH solution (Reagent E)
0.6 ml Distilled water
2. Equilibrate at 37°C for about 5 min.
3. Add 0.03 ml of sample and mix.
4. Record the increase of absorbance at 340 nm in a spectrophotometer thermostated at 37°C, and calculate the ∆Aper min using the linear portion of the curve (∆AS).
The blank solution is prepared by adding enzyme dilution buffer (Reagent F) instead of sample (∆A0).
Calculation
Activity can be calculated by using the following formula:
6.2: Millimolar extinction coefficient of NADPH at 340 nm (cm2/μmol)
df: Dilution factor
C: Content of β-phosphoglucomutase preparation in sample (mg/ml)
APPLICATIONS
The enzyme is useful for the determination of α-amylase and inorganic phosphate in clinical analysis.
EXPERIMENTAL DATA
