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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 2×1012, 7×1013, 7×1014M 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.
Moscow University Chemistry Bulletin.
2012, Vol. 53, No. 1, P. 12
   

Copyright (C) Chemistry Dept., Moscow State University, 2002
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