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Luciferase from Indian firefly Luciola praeusta (Coleoptera: Lampyridae: Luciolinae) was isolated and the properties compared with that of the North American firefly, Photinus pyralis. Luciola praeusta luciferase was purified using acetone extraction, gel-filtration column chromatography, ammonium sulfate precipitation and anion exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a homogeneous preparation and the molecular mass was slightly higher than that of Photinus pyralis. The effect of pH, buffer composition and metal ions on the spectral characteristics was studied. The maximum bioluminescence activity of luciferase was observed in ACES buffer at pH 6.5. The emission maximum of 562 nm (in crude extract) was red shifted to 570 nm in Tricine buffer at pH 7.8. In addition, the effect of bovine serum albumin on the storage stability of the protein was investigated. Based on the unique spectral characteristics observed, we propose that Luciola praeusta luciferase in the native form is suitable for the assay of biochemical metabolites in acidic pH.

The quenching of 2,5-diphenyloxazole (PPO) fluorescence by nucleotides has been investigated by electronic absorption and steady state fluorescence spectra. Five purine nucleotides AMP, ADP, ATP, GMP and dGMP, one pyrimidine nucleotide UMP and one dinucleotide NAD have been employed in the present study. Electronic absorption studies indicate that there is no ground state complexation between the nucleotides and PPO. The quenching of PPO fluorescence was investigated at two different wavelengths. When excited at 304 nm, the lambda(max) of PPO, the fluorescence spectra of PPO is quenched following Stern-Volmer kinetics. The quenching ability of nucleotides are in the order NAD>AMP>ADP>GMP>dGMP>UMP. The K(SV) and k(q) values obtained indicate that AMP is a better quencher of PPO fluorescence than GMP, which is contrary to commonly observed pattern. The quenching is found to be dynamic in nature. However, when excited at 260 nm, the absorption maximum of the nucleotides, the fluorescence intensity of PPO is reduced with increase in the concentration of the nucleotide. This is attributed to the primary inner filter effect arising due to the absorption of the incident radiation by the nucleotides. Thus the inner filter effect phenomenon can be employed to assay the non-fluorescent molecules by fluorimetry.

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