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G. Yu. Lomakina, A. E. Bezrukih, N. N. Ugarova
Effect of gelatin on properties
of Luciola mingrelica firefly luciferase
Abstract
The ATP-reagent, including mutant thermostable Luciola
mingrelica firefly luciferase, luciferin, MgSO4, buffer
components and stabilizers are widely used to assay nano- and picomolar
concentrations of adenosine-5¢-triphosphate
(ATP) in various biological samples. The aim of this study was to estimate
activity, stability and analytical characteristics of the ATP-reagent in water
and gelatin solutions and in gelatin gel. At 5% gelatin, we had ATP-reagent
solution at temperatures ³30°. Below 30°
the gelatin gel was formed, and thin films with ATP-reagent were prepared. So,
the ATP-reagent solutions were studied at 30°, whereas in gelatin films it was
assayed at 22°. The gelatin additives were shown to decrease slightly activity
and stability of luciferase. The sensitivity of ATP assay (over 0.96) did not
depend on gelatin presence and aggregate state of the disperse system. The
detection limits were 2×10–12, 7×10–13, 7×10–14M ATP for ATP-reagent in gelatin films, in
solution in the presence of 5% gelatin and in its absence, correspondently. The
storage of ATP-reagent at 22° in gelatin gel not only conserved the enzyme
activity but protected the enzyme from bacterial contamination.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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