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1.
PLoS One ; 6(5): e20073, 2011.
Article in English | MEDLINE | ID: mdl-21603648

ABSTRACT

Over the past decade, firefly Luciferase (fLuc) has been used in a wide range of biological assays, providing insight into gene regulation, protein-protein interactions, cell proliferation, and cell migration. However, it has also been well established that fLuc activity can be highly sensitive to its surrounding environment. In this study, we found that when various cancer cell lines (HeLa, MCF-7, and 293T) stably expressing fLuc were treated with staurosporine (STS), there was a rapid loss in bioluminescence. In contrast, a stable variant of Renilla luciferase (RLuc), RLuc8, exhibited significantly prolonged functionality under the same conditions. To identify the specific underlying mechanism(s) responsible for the disparate sensitivity of RLuc8 and fLuc to cellular stress, we conducted a series of inhibition studies that targeted known intracellular protein degradation/modification pathways associated with cell death. Interestingly, these studies suggested that reactive oxygen species, particularly hydrogen peroxide (H(2)O(2)), was responsible for the diminution of fLuc activity. Consistent with these findings, the direct application of H(2)O(2) to HeLa cells also led to a reduction in fLuc bioluminescence, while H(2)O(2) scavengers stabilized fLuc activity. Comparatively, RLuc8 was far less sensitive to ROS. These observations suggest that fLuc activity can be substantially altered in studies where ROS levels become elevated and can potentially lead to ambiguous or misleading findings.


Subject(s)
Apoptosis , Luciferases, Firefly/drug effects , Oxidative Stress , Cell Line, Tumor , Humans , Hydrogen Peroxide/pharmacology , Luciferases, Firefly/metabolism , Luminescent Measurements/standards , Luminescent Proteins/drug effects , Reactive Oxygen Species/pharmacology
2.
Arch Biochem Biophys ; 433(2): 454-65, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15581602

ABSTRACT

Effects of cosolvent concentration on activity of fire fly luciferase, alpha-chymotrypsin, and alcohol dehydrogenase from baker's yeast (Saccharomyces cerevisiae) have been studied for several solvents with varying hydrophobicities (logP from +1.0 to -1.65) and polarities (dielectric constant from 7.4 to 109). The inhibitory effect of the cosolvent is examined in light of Frank's classification of solvents into 'typically aqueous (TA)' and 'typically non-aqueous (TNA).' The solvent concentration at which the enzyme activity decreases to half, the C(50) values, for TA solvents such as 1-cyclohexyl-2-pyrrolidinone, 2-butoxyethanol, 1-methyl-2-pyrrolidinone, tetrahydrofuran, t-butanol, and ethanol correlate quite well with their critical hydrophobic interaction concentration, rather than logP, while those for TNA solvents such as acetonitrile, dimethyl formamide, formamide, and dimethyl sulfoxide correlate well with logP. The interactions of TA solvents with proteins appear to be governed mainly by hydrophobic interactions while both hydrophobic and hydrophilic interactions play important role in case of TNA solvents.


Subject(s)
Alcohol Dehydrogenase/chemistry , Chymotrypsin/chemistry , Luciferases, Firefly/chemistry , Luciferases, Firefly/metabolism , Solvents/chemistry , Water/chemistry , Acetonitriles/chemistry , Acetonitriles/pharmacology , Adenosine Triphosphate/metabolism , Alcohol Dehydrogenase/analysis , Alcohol Dehydrogenase/drug effects , Alcohol Dehydrogenase/metabolism , Animals , Buffers , Chromatography, High Pressure Liquid , Chymotrypsin/analysis , Chymotrypsin/drug effects , Chymotrypsin/metabolism , Circular Dichroism , Dimethyl Sulfoxide/chemistry , Dimethyl Sulfoxide/pharmacology , Dimethylformamide/chemistry , Dimethylformamide/pharmacology , Ethanol/chemistry , Ethanol/pharmacology , Ethylene Glycols/chemistry , Ethylene Glycols/pharmacology , Fireflies/enzymology , Formamides/chemistry , Formamides/pharmacology , Furans/chemistry , Furans/pharmacology , Hydrophobic and Hydrophilic Interactions , Kinetics , Luciferases, Firefly/analysis , Luciferases, Firefly/drug effects , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Solvents/classification , Solvents/pharmacology , Temperature , tert-Butyl Alcohol/chemistry , tert-Butyl Alcohol/pharmacology
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