The enzyme is useful for the determination of inorganic phosphate in clinical analysis.
Reaction:
Sucrose + Orthophosphate →D-Fructose + α-D-Glucose 1-phosphate
Properties
Molecular weight: ca. 56 kDa (gel filtration)
Structure: monomer of 56 kDa (SDS-PAGE)
Isoelectric point: 4.6
Michaelis constant: 3.9×10^-2M (sucrose); 6.2×10^-3M (phosphate)
pH Optimum: 7.5 Fig.1
pH Stability: 5.0–8.0 Fig.2
Optimum temperature :40℃ Fig.3
Thermal stability below: 45℃ Fig.4
Stability (liquid form): stable at 37℃ for at least two weeks Fig.5
Stability (powder form): stable at 30℃ for at least two weeks Fig.6
Inhibitors: glucose, glucono-1,5-lactone
Specificity: sucrose (100), maltose (0), starch (0)
Stabilizer: Sucrose
Assay Procedure
Principle
The appearance of NADPH is measured spectrophotometrically at 340 nm.
Reagents
A. Triethanolamine (TEA) buffer, 0.1 M; pH 7.6: Dissolve 1.86 g of triethanolamine hydrochloride in 90 ml of distilled water, adjust to pH 7.6 with 5 N NaOH, and dilute with distilled water to 100 ml.
B. Potassium phosphate buffer, (a) 0.1 M; pH 6.8, (b) 0.05 M; pH 6.8: (a) Mix 0.1 M KH₂PO₄ and 0.1 M K₂HPO₄ to make a pH 6.8 solution. (b) Dilute 0.1 M potassium phosphate buffer (Reagent B (a)) with the same volume of distilled water.
C. Sucrose solution, 0.32 M: 11.0 g of sucrose/100 ml of distilled water.
D. EDTA solution, 9.9 mM: 37 mg of EDTA·Na₂·2H₂O/10 ml of potassium phosphate buffer (Reagent B (b)).
E. NADP⁺ solution, 12 mM: 9.2 mg of NADP⁺·Na/1.0 ml of distilled water.
F. d-Glucose-1,6-diphosphate (G-1,6-P₂) solution, 0.1 mM: 1.0 mg of G-1,6-P₂ cyclohexylammonium salt/10 ml of distilled water.
G. MgCl₂ solution, 1.0 M: 4.06 g of MgCl₂·6H₂O/20 ml of distilled water.
H. α-Phosphoglucomutase (α-PGM) suspension, 2000 U/ml: Crystalline suspension, 10 mg/ml (200 U/mg).
I. Glucose-6-phosphate dehydrogenase (G6PDH) solution: 2000 U/ml in potassium phosphate buffer (Reagent B (b)).
Sample: Dissolve the lyophilized enzyme to a volume activity of 0.8–1.5 U/ml in ice-cold TEA buffer (Reagent A) immediately before measurement.
Preparation
1. Pipette the following reagents into a cuvette (light path: 1cm).
1.5 ml Potassium phosphate bufer (Reagent B (a))
1.5 ml Sucrose solution (Reagent C)
0.03 ml EDTA solution (Reagent D)
0.1 ml NADP⁺ solution (Reagent E)
0.1 ml G-1,6-P₂ solution (Reagent F)
0.05 ml MgCl₂ solution (Reagent G)
0.01 ml α-PGM suspension (Reagent H)
0.01 ml G6PDH solution (Reagent I )
2. Equilibrate at 25°C for about 5 min.
3. Add 0.02 ml of sample and mix.
4. Record the increase of absorbance at 340 nm in a spectrophotometer thermostated at 25°C, and calculate the ΔA per min using the linear portion of the curve (denoted as ΔAs).
The blank solution is prepared by adding TEA buffer (Reagent A) instead of sampleΔA0.
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 sucrose phosphorylase preparation in sample (mg/ml).
EXPERIMENTAL DATA
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