[Optimal conditions for determing ATP of microbial origin]

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Accurate measurement of adenosine triphosphate (ATP) is crucial in biological and chemical analyses.
• Firefly luciferin-luciferase systems offer a sensitive method for ATP detection.
• Endogenous luminescence in extracts can interfere with accurate ATP quantification.

Purpose of the Study:

• To investigate the endogenous luminescence of firefly luciferin-luciferase extract.
• To determine optimal conditions for using the extract in low ATP concentration assays.
• To assess the stability of the luciferin-luciferase solution for practical applications.

Main Methods:

• Studied endogenous luminescence of firefly luciferin-luciferase extract.
• Identified optimal extract concentration to minimize endogenous luminescence.
• Assessed solution stability at room temperature, upon dilution, and after cryopreservation (liquid nitrogen freezing and thawing).
• Quantified ATP content in Sarcina flava cells using the optimized assay after cell disruption via ultrasound.

Main Results:

• An optimal concentration of luciferin-luciferase extract was identified to minimize endogenous luminescence interference.
• Luciferin-luciferase solutions demonstrated instability at room temperature and when diluted.
• Solution activity remained unchanged after freezing in liquid nitrogen and subsequent thawing.
• ATP content was successfully assayed in Sarcina flava cells using the developed method.

Conclusions:

• Optimizing luciferin-luciferase extract concentration is essential for accurate low-level ATP determination.
• Cryopreservation offers a viable method for stabilizing luciferin-luciferase solutions for extended use.
• The developed method allows for reliable ATP quantification in biological samples like Sarcina flava cells.