Isolation and characterization of luciferase from Indian firefly, Luciola praeusta.
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.
JONAKI Laboratory, Board of Radiation and Isotope Technology, CCMB Campus, Hyderabad-, 500007, India; SMARTS, Metallurgy & Materials Group, Indira Gandhi Centre for Atomic Research, Kalpakkam-, 603102, India.
Thuraimony
T
Muthukumaran
Nemani V
NV
KrishnaMurthy
Nagalingam
N
Sivaprasad
Thankiah
T
Sudhaharan
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D001517:Coleoptera / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D008156:Luciferases / Q000737:chemistry / Q000302:isolation & purification*; D008297:Male; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D008156:Luciferases
Bioluminescence; Characterization; Firefly luciferase; Luciola praeusta; Purification