Glutamate dehydrogenase is an enzyme in both prokaryotes and eukaryotic mitochondria. Glutamate dehydrogenase can be used for the enzymatic determination of ammonia, alpha-ketoglutaric acid, L-glutamate and urease.
Application
This enzyme can be used for the enzymatic determination of NH₃, α-ketoglutarate, and L-glutamic acid, and also for the activity detection of leucine aminopeptidase and urease; in clinical analysis, by coupling with urease, it can also be used for the enzymatic determination of urea.
Assay procedure
Principle
The disappearance of NADH is measured at 340nm by spectrophotometry.
Unit definition
One unit (U) is defined as the amount of enzyme which produces one micromole of NAD⁺ per minute at 25℃ and pH 7.3.
Reagents
A. Potassium phosphate buffer, 50mM, pH7.4:Mix 50Mm KH₂PO₄ solution and 50mM K₂HPO₄ solution to make a pH7.4 solution.
B. Imidazole-HCl buffer, 58mM, pH7.3:Dissolve 3.95g of imidazole in 800ml of distilled water, adjust to pH7.3 with 2N HCl and dilute with distilled water to 1000ml.
C. α-Ketoglutarate solution, 0.13M, pH6.0~7.0:Dissolve 475mg of α-ketoglutarate in 8ml of distilled water, adjust to between pH6.0 and 7.0 with 2N NaOH and dilute with distilled water to 25ml.
D. NADH solution, 4mM:14mg of NADH.2Na, (MW=709.4) /5ml of 5mM Potassium phosphate buffer, pH7.4.
E. Ammonium acetate(1.6M)-EDTA(4mM) solution: Dissolve 1.23g of ammonium acetate and 14.9mg of EDTA.2Na in 8ml of distilled water and dilute with distilled water to 10ml.F. Enzyme dilution buffer:Imidazole-HCl buffer(Reagent B) containing 0.1% bovine serum albumin(BSA).
Sample: Dissolve up to 5mg of the lyophilized enzyme/ml in ice-cold potassium phosphate buffer(Reagent A).
Dissolve the enzyme solution to a volume activity of 0.20~0.30U/ml with ice-cold enzyme dilution buffer (Reagent F) immediately before measurement.
Procedure
1. Pipette the following reagents into a cuvette (light path:1cm).2.4ml Imidazole-HCl buffer (Reagent B)0.3ml α-Ketoglutarate solution (Reagent C)0.1ml NADH solution (Reagent D)0.1ml Ammonium acetate-EDTA solution (Reagent E)
2. Equilibrate at 25℃ for about 5 min.
3. Add 0.1ml of sample and mix.
4. Record the decrease of absorbance at 340nm in a spectrophotometer thermostated at 25℃,and calculate the ΔA per min using the linear portion of the curve (ΔAₛ).The blank solution is prepared by adding enzyme dilution buffer(Reagent F) instead of sample(ΔA₀).
Calcuation
Activity can be calculated by using the following formula:
| Vt: | Total volume (3.00ml) |
| Vs: | Sample volume (0.1ml) |
| 6.22: | Millimolar extinction coefficient of NADH under the assay condition（cm²/μmol） |
| 1.0: | Light path length（cm） |
| df: | Dilution factor |
| C: | Enzyme concentration in dissolution（c mg/ml） |
