A rapid assay method for ammonia using glutamine synthetase from glutamate-producing bacteria

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Ammonia determination is crucial in various fields, including clinical diagnostics and environmental monitoring.
• Existing enzymatic assays for ammonia, often relying on glutamate dehydrogenase, can be time-consuming.

Purpose of the Study:

• To develop a rapid and sensitive enzymatic assay for ammonia determination.
• To compare the efficiency of the new assay with existing methods.

Main Methods:

• Utilized glutamine synthetase, pyruvate kinase, lactate dehydrogenase, and NADH for a rapid enzymatic assay.
• Modified the assay for spectrophotometry in the visible region using pyruvate oxidase, peroxidase, and chromogens.
• Applied the assay to determine ammonia in serum, microbial culture broth, and wastewater.

Main Results:

• The glutamine synthetase-based assay determined 25 nmol of ammonia in 5 minutes, significantly faster than glutamate dehydrogenase methods (14-30 minutes).
• Spectrophotometric modifications enhanced sensitivity, achieving 0.65 and 1.7 times the sensitivity of glutamate dehydrogenase assays with different chromogens.
• The assay successfully detected ammonia in diverse samples and was adaptable for continuous monitoring of ammonia-forming enzymes and ATP-ADP determination.

Conclusions:

• A rapid, sensitive, and versatile enzymatic assay for ammonia determination has been established using glutamine synthetase.
• This method offers significant advantages in speed and adaptability over traditional assays.
• The assay has broad applicability in clinical, environmental, and biochemical analyses.