Effect of gelatin on properties of Luciola mingrelica firefly luciferase
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An ATP-reagent containing thermostable mutant of Luciola mingrelica firefly luciferase, MgSO4, components of buffer solution, and stabilizers, which is widely used to detect nano- and picomolar concentrations of adenosine-5′-triphosphate (ATP) in various biological samples, is been object of this study. The activity, stability, and analytical characteristics of the ATP-reagent have been assessed in the presence and absence of 5% gelatin and in gelatin gel. The solution of ATP-reagent was obtained at 30°C and a gelatin concentration of 5%, while gel formation occurred at 22°C. The gelatin addition decreased the activity and stability of luciferase slightly. The sensitivity of ATP detection (above 0.96) did not depend on the gelatin presence and the aggregate state of the disperse system. Limits of detection were 2 × 10−12, 7 × 10−13, and 7 × 10−14 M ATP, when the ATP-reagent was used in gelatin films and in the solution in the presence of 5% gelatin and in the absence, respectively. It was shown that the storage of ATP-reagent in gelatin gel not only preserved enzymatic activity, but protected the enzyme from bacterial contamination, which was the cause of the enzyme activity loss.
Keywordsfirefly luciferase Luciola mingrelica ATP-reagent stability thermal inactivation gelatin gel
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