SPECIFICATION

PROPERTIES

APPLICATIONS

The enzyme is useful for the determination of L-glutamine in clinical analysis.

ASSAY PROCEDURE

Principle

The appearance of NADH is measured spectrophotometrically at 340 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of NADH per min at 37℃ and pH 6.0 under the conditions described below.

Reagents

1. L-Glutamine solution, 2.0%: 4.0 g of L-glutamine/200 ml of distilled water.
2. Acetate buffer, 0.2 M; pH 6.0: mix 0.2 M acetic acid solution and 0.2 M sodium acetate solution to make a pH 6.0 solution.
3. Perchloric acid solution, 0.75 N: 25 ml of perchloric acid (60%)/200 ml of distilled water.
4. Sodium hydroxide solution, 1.5 N: 12 g of sodium hydroxide/200 ml of distilled water.
5. Hydroxylamine buffer, 0.25 M; pH 8.0, containing 20 mm EDTA: dissolve 3.48 g of hydroxylamine hydrochloride and 1.49 g of EDTA·Na₂·₂H₂O in 160 ml of distilled water, adjust to pH 8.0 with 2 N KOH and dilute with distilled water to 200 ml.
6. NAD⁺ solution, 10 mM: 1.33 g of NAD⁺/200 ml of distilled water.
7. Tris-HCl buffer, 50 mm; pH 7.5, containing 0.1 mm EDTA: dissolve 1.21 g of Tris(hydroxymethyl)aminomethane and 7.4 mg of EDTA·Na₂·₂H₂O in 160 ml of distilled water, adjust to pH 7.5 with 2 N HCl and dilute with distilled water to 200 ml.
8. Glutamate dehydrogenase (GLDH) solution, 770 U/ml: mix 33 ml of Tris-HCl buffer (Reagent G) and 41.3 g of glycerol, and dissolve 50000 U of GLDH.
9. Enzyme dilution buffer: mix 20 mM acetic acid solution and 20 mm sodium acetate solution to make a pH 6.0 solution.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.5–3.0 U/ml with ice-cold enzyme dilution buffer (Reagent I) immediately before measurement.

Procedure

1. Pipette the following reagents into a test tube.

   0.2 ml	L-Glutamine solution	(Reagent A)
   0.4 ml	Acetate buffer	(Reagent B)

2. Equilibrate at 37℃ for about 5 min.
3. Add 0.2 ml of sample and incubate for 10 min at 37℃.
4. Add 0.2 ml of perchloric acid solution (Reagent C) to stop the reaction.
5. Add 0.1 ml of sodium hydroxide solution (Reagent D) to neutralize the mixture.
6. Collect the supernatant fluid by centrifugation.
7. Pipette successively the following reagents into a test tube.

   1.0 ml	Hydroxylamine buffer	(Reagent E)
   0.5 ml	NAD+ solution	(Reagent F)
   1.0 ml	Distilled water
   0.05 ml	The supernatant of step 6
   0.025 ml	GLDH solution	(Reagent H)

8. Incubate for 30 min at 37℃.
9. Read the absorbance at 340 nm in a cuvette (light path: 1 cm) (A_S).
   The blank solution is prepared by reversing the sequence of sample and perchloric acid solution (Reagent C) (A₀).

Calculation

Activity can be calculated by using the following formula:

6.2 : Millimolar extinction coefficient of NADH at 340 nm (cm²/μmol)
df : Dilution factor
C : Content of glutaminase preparation in sample (mg/ml)

EXPERIMENTAL DATA

FAQ

REFERENCES

Roberts, E., “The Enzymes,” Vol. 4 (2nd ed.), Academic Press, New York and London, 1960, pp. 285–300.

Hartman, S. C., “The Enzymes,” Vol. 4 (3rd ed.), Academic Press, New York and London, 1971, pp. 79–100.

Ayodeji A. et. al. (2019) Recent advances in microbial glutaminase production and applications—a concise review, Crit. Rev. Biotechnol., 39, 944-963