仕様

特性

アプリケーション

The enzyme is useful for the determination of α-amylase and inorganic phosphate in clinical analysis.

分析手順

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 converts 1 μmol of β-d-glucose-1-phosphate to β-d-glucose-6-phosphate per min at 37℃ and pH 7.0 under the conditions described below.

Reagents

1. 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.
2. 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.
3. NADP⁺ solution, 12 mM: 230 mg of NADP⁺·Na/25 ml of distilled water.
4. 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.
5. Glucose-6-phosphate dehydrogenase (G6PDH) solution: 1750 U/ml.
6. 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)
   0.03 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℃ 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℃, and calculate the ΔA per min using the linear portion of the curve (ΔA_S).
   The blank solution is prepared by adding enzyme dilution buffer (Reagent F) 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 β-phosphoglucomutase preparation in sample (mg/ml)

実験データ

FAQ

参考文献

• Nakamura, K. et al., J. Ferment. Bioeng., 85, 350–353 (1998).
• Shirokane, Y. et al., Carbohydr. Res., 329, 699–702 (2000).