Glutamate dehydrogenase (GLDH-R)

Enzymes for Clinical Chemistry

The enzyme is useful for BUN (blood urea nitrogen) test and elimination of free ammonia.

Origin	microorganism
Systematic name	L-Glutamate: NADP⁺ oxidoreductase (deaminating)
EC Number	1.4.1.4
Reaction formula	L-glutamate + H₂O + NADP⁺ = 2-oxoglutarate + NH₃ + NADPH + H⁺

SPECIFICATION

Appearance	white to light yellow lyophilizate
Activity	≧10.0 U/mg
Contaminant	Catalase　≦ 0.5 U/U%
Stabilizer	trehalose
Storage condition	below -20℃

PROPERTIES

Molecular weight	ca. 49 kDa (gel filtration)
Michaelis constant	1.97mM (Glutamate) 0.16mM (NADP⁺)
pH Optimum	9.0 (Fig. 1)
pH Stability	7.0‐10.0 (Fig. 2)
Optimum temperature	35‐40°C (Fig. 3)
Thermal stability	below 40°C (Fig. 4)
Stability (powder form)	stable at 37°C for at least one week

APPLICATIONS

The enzyme is useful for BUN (blood urea nitrogen) test and elimination of free ammonia.

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 37℃ and pH 9.0 under the conditions described below.

Reagents

0.1M L-Glutamate / 0.1M Pyro-phosphate sodium salt hydrate:dissolve 2.94 g of L-Glutamate and 8.92 g of Pyro-phosphate sodium salt hydrate in 180 ml of distilled water, adjust to pH 9.0 ± 0.05 with 1N NaOH and dilute with distilled water to 200ml.
10 mM β-NADP⁺ solution: dissolve 76.5mg of β-NADP⁺ sodium salt in 10 mL distilled water.
Enzyme dilution buffer: dissolve 3.58 g of Disodium hydrogen phosphate, 0.24 g of Potassium dihydrogen phosphate and 0.2 g of Potassium chloride, 8.0 g of Sodium chloride in 800 mL distilled water, adjust to pH 7.40 ± 0.05 with Hydrogen chloride and dilute with distilled water to 1000 mL.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.25 - 0.70 U/mL in enzyme dilution buffer (Reagent C) immediately before measurement.

Procedure

Pipette the following reaction mixture immediately before use.

2.75 mL 0.1 M L-Glutamate/ 0.1M Pyro-phosphate solution (Reagent A)

0.15 mL β-NADPH solution (Reagent C)
Equilibrate at 25℃ for about 5 min.
Add 0.10 mL of sample and mix gently.
After 15 seconds, Record the increase of absorbance at 340 nm in a spectrophotometer thermostated at 25℃, and calculate the ΔA_S per min from 1 minute to 2 minutes.
The blank solution is prepared by adding enzyme dilution buffer (Reagent C) instead of sample (A₀).