SPECIFICATION

PROPERTIES

APPLICATIONS

The enzyme is useful for the determination of inorganic phosphate in clinical analysis.

ASSAY PROCEDURE

Principle

The appearance of NADPH is measured spectrophotometrically at 340 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of NADPH per min at 25℃ and pH 6.8 under the conditions described below.

Reagents

1. 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.
2. 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 same volume of distilled water.
3. Sucrose solution, 0.32 M: 11.0 g of sucrose/100 ml of distilled water.
4. EDTA solution, 9.9 mM: 37 mg of EDTA·Na₂·2H₂O/10 ml of potassium phosphate buffer (Reagent B (b)).
5. NADP⁺ solution, 12 mM: 9.2 mg of NADP⁺·Na/1.0 ml of distilled water.
6. 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.
7. MgCl₂ solution, 1.0 M: 4.06 g of MgCl₂·6H₂O/20 ml of distilled water.
8. α-Phosphoglucomutase (α-PGM) suspension, 2000 U/ml: crystalline suspension, 10 mg/ml (200 U/mg).
9. Glucose-6-phosphate dehydrogenase (G6PDH) solution: 2000 U/ml of 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.

Procedure

1. Pipette the following reagents into a cuvette (light path: 1 cm).

   1.5 ml	Potassium phosphate buffer	(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℃ for about 5 min.
3. Add 0.02 ml of sample and mix.
4. Add 0.1 mL of sample and mix.
5. Record the increase of absorbance at 340 nm in a spectrophotometer thermostated at 25℃, and calculate the ΔA per min using the linear portion of the curve (ΔA_S).
   The blank solution is prepared by adding TEA buffer (Reagent A) instead of sample (ΔA₀).

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

REFERENCES

• Koga, T. et al., Agric. Biol. Chem., 55, 1805–1810 (1991).
• Kitao, S. and Nakano, E., J. Ferment. Bioeng., 73, 179–184 (1992).