SPECIFICATION

PROPERTIES

APPLICATIONS

The enzyme is useful for the determination of ammonia and ATP in clinical analysis.

ASSAY PROCEDURE

Principle

The appearance of phosphate is measured spectrophotometrically at 660 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of phosphate per min at 37℃ and pH 7.0 under the conditions described below.

Reagents

1. Imidazole–HCl buffer, 1.0 M; pH 7.0: dissolve 6.81 g of imidazole in 80 ml of distilled water, adjust to pH 7.0 with 4 N HCl and dilute with distilled water to 100 ml.
2. ATP solution, 0.12 M: dissolve 0.726 g of ATP·Na₂·3H₂O in 8 ml of distilled water, adjust to pH 7.0 with 1 N NaOH and dilute with distilled water to 10 ml.
3. L-Glutamate solution, 2.0 M: dissolve 37.4 g of sodium l-glutamate in 80 ml of distilled water, adjust to pH 7.0 with 1 N NaOH and dilute with distilled water to 100 ml.
4. NH₄Cl solution, 1.0 M: 5.35 g of NH₄Cl/100 ml of distilled water.
5. MgCl₂ solution, 1.67 M: 34.0 g of MgCl₂·6H₂O/100 ml of distilled water.
6. FeSO₄ solution, 29 mM: 0.8 g of FeSO₄·7H₂O/100 ml of 0.3 N H₂SO₄ (prepare freshly).
7. Ammonium molybdate reagent, 53 mM: 6.6 g of (NH₄)₆Mo₇O₂₄·4H₂O/100 ml of 7.5 n H₂SO₄.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.08–0.18 U/ml in ice-cold 50 mm imidazole–HCl buffer, pH 7.0, immediately before measurement.

Procedure

1. Prepare the following reaction mixture.

   1.0 ml	Imidazole–HCl buffer	(Reagent A)
   2.5 ml	ATP solution	(Reagent B)
   2.0 ml	L-Glutamate solution	(Reagent C))
   1.0 ml	NH4Cl solution	(Reagent D)
   0.6 ml	MgCl2 solution	(Reagent E)
   2.9 ml	Distilled water

2. Pipette 0.2 ml of the reaction mixture and 0.1 ml of distilled water in a test tube.
3. Equilibrate at 37℃ for about 5 min.
4. Add 0.1 ml of sample and incubate for 10 min at 37℃.
5. Add 1.8 ml of FeSO₄ solution (Reagent F) to stop the reaction, and then add 0.15 ml of ammonium molybdate reagent (Reagent G).
6. Read the absorbance at 660 nm in a cuvette (light path: 1 cm) (A_S).
   The blank solution is prepared by reversing the sequence of the addition of sample and FeSO₄ solution (Reagent F)(A₀).

Calculation

Activity can be calculated by using the following formula:

0.556 : Millimolar extinction coefficient of phosphate under the assay conditions (cm²/μmol)
df : Dilution factor
C : Content of glutamine synthetase preparation in sample (mg/ml)

EXPERIMENTAL DATA

FAQ

REFERENCES

Ebner, E. et al., “Methods in Enzymology,” Vol. 17A, Academic Press, New York, 1970, pp. 910–922.

David E. et al.(2000) Structure–function relationships of glutamine synthetases, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1477, 122-145