SPECIFICATION

PROPERTIES

APPLICATIONS

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

ASSAY PROCEDURE

Principle

The appearance of d-glucose is measured spectrophotometrically at 505 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of d-glucose per min at 30℃ and pH 7.0 under the conditions described below.

Reagents

1. HEPES–NaOH buffer, 50 mM; pH 7.0: dissolve 2.38 g of HEPES in 160 ml of distilled water, adjust to pH 7.0 with 1 N NaOH and dilute with distilled water to 200 ml.
2. Maltose solution, 0.2 M: 3.60 g of maltose monohydrate/50 ml of HEPES–NaOH buffer (Reagent A).
3. Phosphate solution, 0.2 M: dissolve 1.36 g of KH₂PO₄ in 40 ml of HEPES–NaOH buffer (Reagent A), adjust to pH 7.0 with 4 N NaOH and dilute with Reagent A to 50 ml.
4. HCl solution, 5 N: 43 ml of concentrated HCl/100 ml of distilled water.
5. pH Adjusting solution (NaOH solution, 1 N): 4.0 g of NaOH/100 ml of distilled water.
6. Glucose assay kit: “GLUCOSE C II-TEST_WAKO” (Wako Pure Chemical), or a similar glucose assay kit.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.15–0.55 U/ml with ice-cold HEPES–NaOH buffer (Reagent A) immediately before measurement.

Procedure

1. Pipette the following reagents into a test tube.

   0.2 ml	HEPES–NaOH buffer	(Reagent A)
   0.1 ml	Maltose solution	(Reagent B)
   0.1 ml	Phosphate solution	(Reagent C)

2. Equilibrate at 30℃ for about 5 min.
3. Add 0.1 ml of sample and incubate for 10 min at 30℃ [test].
4. The blank solution is prepared by adding HEPES–NaOH buffer (Reagent A) instead of sample [blank].
5. Add 0.1 ml of HCl solution (Reagent D) to stop the reaction.
6. Add 0.5 ml of pH adjusting solution (Reagent E).
7. Pipette 0.3 ml of the test and blank mixture into respective test tubes.
8. Add 3.0 ml of glucose assay kit (Reagent D) and incubate for about 5 min at 37℃.
9. Read the absorbance at 505 nm in a cuvette (light path: 1 cm) [test: A_S, blank: A₀].

Calculation

Activity can be calculated by using the following formula:

6.24 : Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm²/μmol)
1/10 : A factor for correction of the reaction volume
df : Dilution factor
C : Content of maltose phosphorylase preparation in sample (mg/ml)

EXPERIMENTAL DATA

FAQ

REFERENCES

• Hiruma, M. et al., Nippon Nogeikagaku Kaishi, 70, 773–780 (1996).
• Shirokane, Y. et al., Carbohydr. Res., 329, 699–702 (2000).