Firefly Bioluminescence Research Articles

Co‐determination of ATP and proteins in Triton X 100 non‐ionic detergent‐opened monolayer cultured cells

Human monolayer cells (HEp-2 and Hep G2) were cultured in 96-well plates. A modified Triton X 100 nonionic detergent extraction method was used for releasing intracellular ATP and protein in one step. The detergent technique was compared to perchloric acid (PCA) extraction. ATP was determined by the firefly bioluminescence method and ATP values were referred to cell protein (ATP:protein ratio). There was no significant difference in ATP data between detergent and PCA treatments. The ATP:protein ratio seems to be a sensitive tool for characterizing the metabolic activity of monolayer tissue culture cells. The protein-mobilizing capability of Triton X 100 depends on the type of cell culture used. Our modified extraction gives reliable ATP:protein values with one simple extraction step.

The Influence of Insertion of a Critical Residue (Arg356) in Structure and Bioluminescence Spectra of Firefly Luciferase

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.

Dye induced quenching of firefly luciferase–luciferin bioluminescence

The quenching of firefly bioluminescence (BL) in presence of xanthene dyes and tetratolylporphyrin was investigated. The BL intensity was quenched with an altered decay pattern in presence of xanthene dyes and tetratolylporphyrin. The electronic absorption spectra indicate that there is no significant interaction occurring between the dyes and the BL components in the ground state. The BL quenching decay rate and fluorescence quenching studies of luciferin by the dyes suggest an energy transfer through an exciplex, involving oxyluciferin, in the excited state and the dyes, in the ground state. The bimolecular quenching rate constant ( K q) values obtained from fluorescence studies varied between 7.7 × 10 12 and 19.8 × 10 12 M −1 s −1. The magnitude of the bimolecular quenching rate constants confirmed the complex formation between dye and excited oxyluciferin. The exciplex subsequently undergoes a non-radiative decay to the ground state via a combination of heavy atom induced and Förster-type energy transfer. The decay rate constants in presence and in absence of dyes vary between 7.47 × 10 −4 and 7.6 × 10 −2 s −1. In the presence of dyes the effective decay rate constants ( k eff) increased while the lifetime of light emitting species decreased. The kinetic studies in presence of singlet oxygen scavengers, like β-carotene and NaN 3, prove that there is no significant quenching of the firefly BL due to the formation of singlet oxygen.

Estimation of the contribution of the bioluminescent reaction rate and quantum yield to the enhancement of firefly bioluminescence in the presence of cationic liposomes

Cationic liposomes containing phosphatidylcholine, cholesterol and distearyldimethylammonium chloride (DSDAC) enhanced maximum light emission (BL intensity) and total light emission from the firefly bioluminescence (BL) reaction. The increase in BL intensity was interpreted on the basis of the increase in both BL reaction rate and BL quantum yield (PhiBL) of the BL reaction. The increase in BL reaction rate was due to the increase in the localized concentration of BL reactants on the surface of cationic liposomes by electrostatic interaction. On the other hand, the increase in PhiBL was due to the change of light-emitting species in the presence of cationic liposomes. Each contribution of BL reaction rate and PhiBL to the enhancement of the BL intensity was estimated by measuring the BL reaction rate and PhiBL in the presence of cationic liposomes containing various amounts of DSDAC. The contribution of the BL reaction rate to the increase in the BL intensity was found to be two-fold greater than that of PhiBL.

Noninvasive indirect imaging of vascular endothelial growth factor gene expression using bioluminescence imaging in living transgenic mice

Vascular endothelial growth factor (VEGF) plays a critical role in the early activation of stromal tissues during wound healing and tumor growth. We report the use of a two-step transcriptional amplification (TSTA) approach to augment the transcriptional activity of the relatively weak VEGF promoter (pVEGF) using firefly luciferase (fl) reporter gene and bioluminescence imaging (BLI). In cell culture, we demonstrate that TSTA-based fl gene expression can be significantly enhanced over the direct one-step system. Using a transgenic mouse model (pVEGF-TSTA-fl), we demonstrate the induction of VEGF gene expression using a wound-healing model and a subcutaneous mammary tumor model. In skin-wounding experiments, pVEGF-induced fl expression in the wound lesion is detected on days 4 and 5 and peaks on days 15-22. Furthermore, the bioluminescence signal shows good correlation with the endogenous VEGF protein levels in the wound tissue (r2 = 0.70). In the mammary tumor model, fl expression is detected on day 3, peaks at day 17, and declines thereafter. These results support the use of noninvasive BLI for the longitudinal monitoring of VEGF induction during wound healing and tumor progression, and this mouse model should find use in various applications in which it is important to noninvasively study VEGF gene expression.

Construction of a new firefly bioluminescence system using l-luciferin as substrate

l-Luciferin can be converted into d-luciferin with an enzyme/co-factor system consisting of firefly luciferase, an esterase, ATP, Mg 2+, and coenzyme A. By this means, a new firefly bioluminescence system can be constructed that uses l-luciferin as the substrate.

Bioluminescence determination of enzyme activity of firefly luciferase in the presence of pesticides

Firefly luciferase (EC 1.13.12.5) (FL) is the key enzyme in the firefly bioluminescence method (FB), which is widely used to determine the viability of living cells. The FB method can also be applied to monitoring the influence of different pollutants, such as pesticides. Firefly luciferase is a hydrophobic enzyme and its activity depends on the type of solvent, pH and substances present in the reaction mixture. The influence of three aromatic pesticides, including fenoxaprop-p-ethyl (I), diclofop-methyl (II) and metsulfuron methyl (III), on the enzyme activity was indirectly evaluated through the measurement of emitted light in the bioluminescence reaction, expressed in relative luminescence units (RLU). The reaction mixture used in the bioluminescence measurements consisted of: Tris buffer (pH 7.75), adenosine triphosphate (ATP) and ATP monitoring reagent, where FL is present. Ethanol-water solutions of each pesticide were then added at concentrations of 2.4 x 10(-4)-2.4 x 10(-8) mol/L. The FL activity inhibition factors (FL In%) were determined. The FL activity was maximally inhibited in the presence of all pesticides under study at a concentration of 2.4 x 10(-4) mol/L and was lowered by about 15-26% for pesticide I at concentrations of 2.4 x 10(-5)-2.4 x 10(-8) mol/L, whereas pesticides II and III, applied in the same concentration range, showed smaller FL inhibition values (5.3-20%). The pesticide degradation products (obtained after a 1 month period), measured in the same experimental conditions, in most cases exhibited a much less inhibitory effect on the enzyme activity than the corresponding initial pesticide.

Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence Are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain

Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866-2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved beta-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent beta-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.

Exogenous compounds in studying the mechanism of electron-excited state formation in bioluminescence.

Using a series of exogenous fluorescent molecules as potential energy acceptors, the hypothesis on the activity of the upper electron-excited states in bioluminescence was tested. The results in bacterial and firefly bioluminescent enzyme systems were compared. Similar activity to the energetic precursor in bacterial bioluminescence was not proven in the case of the firefly system, the result of a very efficient intramolecular energy transfer in the emitter of the firefly bioluminescence. The influence of a number of metallic salts on a bacterial bioluminescent enzyme system was studied. Bioluminescence inhibition coefficients were compared to the free energies of electron withdrawing of cations. The correlation shows that inhibition and activation of luminescence intensity result from the effects of cations on electron transfer in the bioluminescent system.

Luminescence literature 2000. Firefly bioluminescence.

AbstractThe journal provides comprehensive literature searches on all aspects of luminescence which will be published in most issues. These lists have been produced regularly since 1986—some by year and some by specialized topic. This survey of papers on Firefly and marine bacterial bioluminescence is drawn from the 2000 literature and covers the biology of these organisms and the applications of genes and proteins from these organisms in analysis.

Rapid and specific detection of bacteria using bioluminescence

This paper describes rapid methods for the detection of very low numbers of bacteria. Specificity was obtained by the use of antibodies in immunomagnetic separation, a bacteriophage to allow targeted cell lysis, or a combination of both. The amplified endpoint assay used cell-derived adenylate kinase to convert added adenosine diphosphate (ADP) to adenosine triphosphate (ATP) which could then act as a substrate for the firefly bioluminescence reaction. Specific and non-specific assays were evaluated using Escherichia coli O157 as the test organism. Limits of detection of around 10 2 cells ml −1 could be obtained with an upper end cut-off of around 10 7 cells ml −1. Depending on the level of specificity offered, the assays took from 5 min to just under 1 h.

From the firefly bioluminescence to the dioxetane-based (AMPPD) chemiluminescence immunoassay: a retroanalysis

Chemiluminescent dioxetanes, which are enzymically triggerable, have proved to be a valuable tool in diagnostic clinical applications. In this context, the phenomenon of firefly bioluminescence (the most efficient light-producing system so far) has served as an excellent example to develop new chemiluminescent probes for bioanalysis, in particular chemiluminescence immunoassays. From the recognition of the molecular details of the firefly bioluminescence, a chemical equivalent has been conceived which possesses the essential features for efficient generation of light, i.e., thermal persistence through spiroadamantyl substitution, spontaneous chemically initiated electron exchange luminescence (CIEEL)-active decomposition after enzymic triggering, efficient light emission as a lasting glow and convenient synthesis by photooxygenation. This has led to a rationally designed dioxetane, namely the 3-(2′-spiroadamantyl)-4-methoxy-4-(3″-phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) derivative, which serves as a substrate for alkaline phosphatase and, in terms of sensitivity, has surpassed the hazardous radioactive immunoassay probes. This paper gives a historical account in the form of a retroanalysis of the success story behind the rational design of the first enzymically triggerable, CIEEL-active AMPPD chemiluminescent probe.

Sensitive Analysis of Protein Phosphatase Inhibitors by the Firefly Bioluminescence System: Application to PP1γ

Protein phosphatases are classified into types 1 (PP1) and 2 (PP2A, 2B, and 2C). We have already established a new analysis method for PP2A inhibitors by using the firefly bioluminescence system for detection. This method was successfully applied to determine the PP1γ inhibitory activity of known inhibitors, i.e., calyculin A, microcystin-LR, and tautomycin, requiring less than 10 pmol of a sample.

New Analysis Method for Protein Phosphatase Type 2A Inhibitors Using the Firefly Bioluminescence System

A new analysis method for protein phosphatase type 2A inhibitors was established that uses the firefly bioluminescence system for detection. Thus, firefly luciferin phosphate was used as a substrate, and the liberated free luciferin was determined from the amount of light emitted from the immobilized luciferase. This method was successfully used to determine the activities of known inhibitors, i.e., okadaic acid, calyculin A, microcystin-LR and tautomycin using less than 10 pmol of a sample.

Determination of validity for blood for transfusion by firefly bioluminescent assay for ATP

The duration for which blood used in transfusion is acceptable is an important factor. The validity of blood, conserved and kept at 4°C, has been established by ATP measurement (mol l −1) using a rapid firefly bioluminescence method. ATP is extracted from the blood cells with trichloroacetic acid, diluted with Tris buffer (pH 7.75). Samples conserved with CPDA-1 and ACD were compared. Blood stabilised with CPDA-1 was valid for transfusion until the 33rd day, that stabilised with ACD until the 21st day.

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable.

An analytical procedure for the measurement of ATP extracted from activated sludge

There has been numerous attempts to link the ATP content of activated sludge to certain wastewater characteristics or to certain control parameters used in the operation of wastewater treatment plants. Published results vary and result from the use of different ATP extraction and measurement methods. This study deals with the development of an analytical method that requires filtration on polycarbonate membrane (0.4 μm) and a subsequent ATP extraction with DMSO. The filtration step overcomes problems of enzymatic inhibition in the firefly bioluminescence assay due to dissolved substances in the liquid fraction of the activated sludge samples. In comparison with extraction in boiling TRIS buffer, the most widely used method, extraction in DMSO was found to be effective and advantageous owing to its simplicity and reliability, the ease of its execution, and its potential use in routine measurements. The application of this analytical procedure to aerobic sludges in batch culture yielded a higher estimate of the average value of the ATP pool than previously published results.

Measurement of the adenylate energy charge in Nereis diversicolor and Nephtys sp. (Polychaeta: Annelida):

ATP-content and adenylate energy charge (AEC) ratios were determined in two polychaete species (Neries diversicolor and Nephytys sp.), sampled in ten stations along the heavily polluted Western Scheldt estuary (N. Belgium, S. Holland). The samples were taken between 27 December 1982 and 6 January 1983. Nereis diversicolor was also sampled in an unpolluted brackish water pond, and subjected to artificial stress by drying the organisms on filter paper. Adenine nucleotide levels were determined using the firefly bioluminescence reaction. The light emission was measured with the integration mode. As the “Constant Light Signal” reagent used contained AMP and probably ADP itself, both myokinase and pyruvatekinase had to be inactivated before the assay. The use of pepsin in acidic conditions to denaturate these enzymes is discussed. For both N. diversicolor and Nephtys sp., a significant relation between ATP-content and fresh weight is found in most samples. Within each species, ATP-content per g fresh weight is dependent on the sampling method, but it is not significantly different between stations. It is higher in Nephtys sp. from the W. Scheldt (130. 8 10-5 g ATP/g fresh weight) than in N. diversicolor from the Dievengat (83.9 10-5 g ATP/g fresh weight). This value in turn is significantly higher than that for N. diversicolor in the W. Scheldt (64.0 10-5 g ATP/g fresh weight). In the drying experiment, AEC gradually decreases with increasing drying time. In the field, AEC is generally high and not significantly different between the three populations. AEC ratios also do not differ between stations within each population. It is argued that field monitoring of AEC in invertebrates is not valuable as a pollution assessment tool for two reasons. In polluted stations, only pollutant-resistant species will be found, and there is evidence that the maintenance of a stable population is impossible when the individuals constantly have low AECs.

Adenosine 5'-triphosphate content of Streptococcus mutans GS-5 during starvation in a buffered salt medium.

Adenosine 5′-triphosphate (ATP) of Streptococcus mutans GS-5 was studied by the firefly bioluminescence method during starvation in a buffered salt medium. There was a decrease in the cellular ATP content with time. The rate of decrease was significantly greater at 37 °C than at 22 °C. The loss in ATP was not accompanied by a decrease in cell number. This variation may lead to errors in the estimation of viable plaque mass by ATP measurement.

Rapid bioluminescent assay for determining netilmicin and tobramycin concentrations in serum.

A rapid bioluminescent assay for determining netilmicin and tobramycin concentrations in serum based on the dose-dependent effect of these agents on the accumulation of extracellular ATP in Escherichia coli LU 14 cultures is presented. This strain of Escherichia coli is unaffected by antibiotics used in combination with aminoglycosides and it lacks significant ATP-ase activity, which is a prerequisite for extracellular ATP accumulation. ATP was quantified by the firefly bioluminescence system. The accuracy of the bioluminescent assay expressed as mean coefficient of variation over the therapeutic range was 3.2%; corresponding figures for EMIT and an agar disk diffusion assay were 4.2% and 4.8% respectively. All methods used correlated well (r = 0.935-0.986) when they were evaluated on clinical serum specimens. The bioluminescent assay requires 25 microliters serum and results are available within 75 min.