Kinetics of the light flash of the living firefly: a supramolecular process.

The light intensity vs. time curve of the light flash of the living firefly has been measured. Unlike the purified firefly enzyme system in aqueous solution, the living system does not show light decay conforming to a double exponential time curve, to simple first or second order decay, or to solid-state Elovich kinetics. Light decay of the living flash does show linearity in a probit vs. square root of time plot, which may indicate a reaction rate-limited by cooperative interactions of a biological phase transition. The observation that the kinetics of the firefly light system differ in the living cell from those in the purified system suggests that in the living system supramolecular factors control the rate of the reaction.     

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.     

The effect of detergents on firefly luciferase reactions

The reaction rate of ATP-limited firefly luciferase-catalysed reactions, is affected by the presence of detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents increase reaction rate several-fold with a sharply defined optimum concentration of detergent for the effect. However, cationic detergents inactivate firefly luciferase during the reaction, resulting in a continuously decreasing reaction rate. Under such conditions, peak light intensity must be used as an indication of initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non-ionic and zwitterionic detergents increase reaction rate over a broad range of detergent concentrations. Enzyme stability during the reaction is not affected by non-ionic detergents and only affected by zwitterionic detergents at high detergent concentration. Cyclodextrins, which can increase reaction rates of some chemiluminescent reactions, have little effect on firefly luciferase activity. Assays for ATP using firefly luciferase must be internally standardized by the constant addition technique in which a known amount of ATP is added to the test sample, since external calibration of such assays, by reference to a previously prepared standard curve, can lead to imprecision when detergents are present.     

A bifunctional luminogenic substrate for two luminescent enzymes: firefly luciferase and horseradish peroxidase.

Horseradish peroxidase (HRP) catalyzes the oxidative chemiluminescent reaction of luminol, and firefly luciferase catalyzes the oxidation of firefly D-luciferin. Here we report a novel substrate, 5-(5'-azoluciferinyl)-2,3-dihydro-1,4-phthalazinedione (ALPDO), that can trigger the activity of HRP and firefly luciferase in solution because it contains both luminol and luciferin functionalities. It is synthesized by diazotization of luminol and its subsequent azo coupling with firefly luciferin. NMR spectral data show that the C5' of benzothiazole in luciferin connects the diazophthalahydrazide. The electronic absorption and fluorescence properties of ALPDO are different from those of its precursor molecules. The chemiluminescence emission spectra of the conjugate substrate display biphotonic emission characteristic of azophthalatedianion and oxyluciferin. It has an optimum pH of 8.0 for maximum activity with respect to HRP as well as luciferase. At pH 8.0 the bifunctional substrate has 12 times the activity of luminol but has 7 times less activity than the firefly luciferin-luciferase system. The specific enhancement of light emission from the cyclic hydrazide part of ALPDO helped in the sensitive assay of HRP down to 2.0 x 10(-13) M and of ATP to 1.0 x 10(-14) mol. Addition of enhancers such as firefly luciferin and p-iodophenol (PIP) to the HRP-ALPDO-H2O2 system enhanced the light output.     

Assay of ATP in intact cells of the facultative phototroph Rhodobacter capsulatus expressing recombinant firefly luciferase

This study reports on the construction, calibration and use of recombinant cells of Rhodobacter capsulatus expressing the luciferase gene of the North American firefly Photinus pyralis to detect, by bioluminescence, variations of endogenous ATP levels under various physiological conditions. We show that the antibiotic polymyxin B allows luciferin to rapidly move into cell cytosol, but does not make external ATP freely accessible to intracellular luciferase. Notably, in toluene:ethanol-permeabilized cells, the apparent K(mATP) for luciferase (50 microM) is similar to that measured in soluble cell fractions. This finding limits the applicability of the firefly luciferase for monitoring intracellular maximal ATP concentration because dark/aerobic-grown recombinant cells of Rba. capsulatus contain approximately 1.3-2.6+/-0.5 mM ATP. Therefore, the effects of chemical and physical factors such as oxygen, light, carbonyl cyanide m-chlorophenyl hydrazone and antimycin A on ATP synthesis were examined in cells subjected to different starvation periods to reduce the endogenous ATP pool below the luciferase ATP saturation level (< or =0.2 mM). We conclude that the amount of endogenous ATP generated by light is maximal in the presence of oxygen, which is required to optimize the membrane redox poise.     

Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase.

This study measured the effect of high pressure on the enzyme kinetics of firefly luciferase. When firefly luciferase is mixed with luciferin and ATP, a transient flash of light is produced, followed by a weak light, lasting hours. The first stage reaction produces an enzyme-luciferin-AMP complex and pyrophosphate. Addition of pyrophosphate to the reaction mixture decelerated the reaction rate, and the initial flash was prolonged to a plateau, showing a quasi-equilibrium state. The effects of temperature and pressure were analyzed at the plateau. The temperature scan showed that the maximum light intensity was observed at about 22.5 degrees C. When pressurized below the temperature optimum, pressure decreased the light intensity, while increasing it above the temperature optimum. According to the theory of absolute reaction rate, the following values were obtained for the bioluminescent reaction: delta V++ = 823.7 - 2.8 T cm3/mol and delta V = -280.47 + 0.94T cm3/mol, where T is the absolute temperature, delta V++ and delta V are, respectively, activation volume and the volume change due to thermal unfolding. The optimal temperature for the maximum light output occurs because the reaction rate increases with the temperature elevation at low temperature range, but the thermal unfolding of the enzyme decelerates the reaction velocity when the temperature exceeds a critical value. The intensity of luminescence is modified by the influence of pressure on both delta V++ and delta V. So long as the volume of the activated complex (V++) exceeds the average volume of the nonactivated complex (VN), pressure will slow down the reaction. At the point where the volumes become equal, there is no change in the rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure will speed up the rate. This study showed that firefly luciferase is not exceptional to other enzymes in responding to high pressure.     

萤火虫的发光行为及其功能起源

发光生物因其活体能自发荧光而倍受关注,萤火虫(鞘翅目:萤科)是被研究最多的发光生物,因为成虫发光在其性选择以及求偶交配中起着重要作用.由于萤火虫的发光是一个消耗能量(ATP),的化学反应过程,因而其发光在成虫之外的虫态也具有重要意义.本文就萤火虫成虫和幼虫的发光行为、功能意义进行了综述,并结合萤火虫的饲养观察对其卵和蛹的发光行为进行了描述,探讨了卵和蛹的形态阶段发光的生物学功能以及生物荧光的起源进化.这将有助于理解萤火虫及其它生物自发荧光的本质和起源进化过程.     

An apparatus for the measurement of very small membrane relaxation currents.

The reaction kinetics of crude firefly lantern extracts with and without added synthetic luciferin were examined. The addition of exogenous luciferin to the reaction mixture resulted in an apparent increase in net light emission per unit of ATP in solution. This additional reactivity (up to 1000-fold) enables the detection of subpicogram levels of ATP. The effects of enzyme preparation, dilution, and aging procedures on the increased sensitivity of the ATP assay, as well as the assay limitations of the crude firefly lantern extracts, are also discussed.     

New application of firefly luciferase--it can catalyze the enantioselective thioester formation of 2-arylpropanoic acid

We introduce a new application of firefly luciferase (EC 1.13.12.7). The firefly luciferases belong to a large superfamily that includes rat liver long-chain acyl-CoA synthetase (LACS1). LACS1 is the enzyme that is involved in the deracemization process of 2-arylpropanoic acid and catalyzes the enantioselective thioester formation of R-acids. Based on the similarity of the reaction mechanisms and the sequences between firefly luciferase and LACS1, we predicted that firefly luciferase also has thioesterification activity toward 2-arylpropanoic acid. From an investigation using three kinds of luciferases from North American firefly and Japanese fireflies, we have confirmed that these luciferases exhibit an enantioselective thioester formation activity and the R-form is transformed to a thioester in preference to the S-form in the presence of ATP, Mg(2+), and CoASH. The enantiomeric excesses of unreacted recovered acid and thioester were determined by chiral phase HPLC analysis and the resulting 2-arylpropanoyl-CoAs were identified by high resolution mass spectroscopy. The K(m) and k(cat) values of thermostable luciferase from Luciola lateralis (LUC-H) toward ketoprofen were determined as 0.22 mM and 0.11 s(-1), respectively. The affinity of ketoprofen was almost the same of d-luciferin. In addition, the calculated E-value toward ketoprofen was approximately 20. These results suggest that LUC-H could catalyze the kinetic resolution of 2-arylpropanoic acid efficiently and would be a new option for the preparation of optically active 2-substituted carboxylic acids.     

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.     

The light of the firefly under the influence of ethyl acetate

When a firefly is made to inhale ethyl acetate vapour, a constant glow appears after a few minutes from its abdominal lantern. This control experiment has been performed by a few workers to record the emission spectrum of the firefly. However, a time-resolved experiment performed by us on this continuous light emitted by the species Luciola praeusta Kiesenwetter 1874 (Coleoptera: Lampyridae: Luciolinae) reveals that it is composed of a continuous train of tiny pulses! The nature of the pulses suggests that an oscillatory chemical reaction continues in the microsecond time scale in the lantern of the anaesthetized firefly.     

Bioluminescence in firefly larvae: A test of the aposematic display hypothesis (Coleoptera: Lampyridae)

We investigated the hypothesis that bioluminescence in firefly larvae (Coleoptera: Lampyridae) functions as an aposematic display. In two experiments, we confirmed the distastefulness of firefly larvae, and tested the hypothesis that a naive, nocturnal predator can learn to use light signals as aposematic cues for avoiding distasteful prey. Larvae were rejected as acceptable prey by 100% of the house mice (Mus musculus) tested. Mice learned to avoid bitter food associated with light cues significantly faster (P=0.003) than mice presented with food lacking light cues. We conclude that luminescent glowing in firefly larvae meets the requirements of an aposematic signal.     

Enhancement of firefly luciferase activity by cytidine nucleotides.

The temporal pattern of light production by firefly luciferase depends on the ATP concentration. With low concentrations of ATP a constant production of light occurred while at high concentrations of ATP (greater than 10 microM) there was a flash of light followed by a decline in light production. This time course of light production with high ATP concentrations was changed from the flash pattern to a pattern with a constant production of light by several cytidine nucleotides. CTP, CDP, dCTP, dCDP, dideoxyCTP, periodate-oxidized CTP and CDP, and the etheno derivatives of CTP and CDP produced that change. CMP, cytidine, CDP-glycerol, CDP-glucose, CDP-ethanolamine, and benzoylbenzoylCTP either were inhibitory to firefly luciferase or were not effective in changing the flash time course. Coenzyme A and related compounds also changed the time course of light production. The changes in time course produced by either cytidine nucleotides or CoA were inhibited by desulfoCoA. These compounds apparently enhanced light production by promoting the dissociation of the inhibitory product, oxidized luciferin, from the enzyme. When the activating compounds were used with high concentrations of ATP, the sensitivity of assay for firefly luciferase was increased. This increased sensitivity is important when using the firefly luciferase gene as a reporter.     

Effect of solvents on the catalytic activity of firefly luciferase

Various solvents stimulate the catalytic activity of firefly luciferase, up to sevenfold. Polyvinylpyrrolidone, polyethylene glycols, and nonionic detergents such as Triton X-100 were the most effective stimulators of the enzyme. Both peak light and total light emission were enhanced in the presence of these solvents indicating an increased turnover of the enzyme. The primary effect of the solvents is on the oxidative reaction rather than the activation reaction. All the experimental data support the hypothesis that the presence of solvent promotes the dissociation of the inhibitory product from the enzyme.     

On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations

A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc.     

Photokinetic microassay of adenylate kinase using the firefly luciferase reaction.

A new rapid photokinetic method is described for determining the activity of adenylate kinase (ATP:AMP phosphotranspherase, EC 2.7.4.3) in 0.1--5.0 micrograms of freeze-dried tissue. This represents a weight range far below that obtainable by fine-needle biopsy. The reaction 2 ADP in equilibrium with AMP + ATP was employed and the ATP formed assayed with firefly luciferase as light yielder. The light emission was recorded on a multi-channel scaler. The adenylate kinase activities found in tissues of mice were in the same range as previously described in a study using fluorometric microassay.     

The role of calcium in fusion of artificial vesicles.

Small phospholipid vesicles (liposomes) fuse upon calcium addition as demonstrated by electron microscopy, light absorbance increases, and mixing of original liposome contents within the boundaries of the fused liposome. The integrity of the fusion event is demonstrated by a novel assay based on the luminescence of firefly extract when mixed with ATP. Subsequent addition of valinomycin or the calcium ionophore A23187 leads to further fusion as shown by electron microscopy, light microscopy, and additional absorbance increase. Concomitant with this second absorbance increase is an increase in the amount of calcium that associates with the liposomes. This increased calcium association is more than can be accounted for by equilibration of 5 mM Ca2+ across the membrane and must indicate exposure of extra calcium binding sites. Binding of calcium to the inner side of the membrane may catalyze the second stage of liposome fusion.     

Two kinetically distinguishable ATP sites in firefly luciferase

Results are presented which indicate that firefly luciferase has two catalytically active sites. One site, Km of 1.1 X 10(-4) M ATP, is responsible for the initial flash and is apparently product inhibited for further light production. The second site, Km of 2 X 10(-5) M ATP, catalyzes the continuous low production of light. ATP or AMP is a potent inhibitor of the initial flash when LH2-AMP is used to initiate the light reaction but appears to have no affect on the second site low level light emission. Both sites must be occupied by ATP for the formation of one L-AMP. Thus, ATP appears to function both as a catalytically active substrate and a regulator for light emission.     

Linear and Non Linear Competition Plots in the Deoxyribose Assay for Determination of Rate Constants for Reaction of non Steroidal Antiinflammatory Drugs with Hydroxyl Radicals

Performing the deoxyribose (DR) assay for determination of the rate constants for reaction of non steroidal antiinflammatory drugs with hydroxyl radicals led to some unusual competition plots. The molecules from the arylpropionic family of drugs: ibuprofen, flurbiprofen, ketoprofen and naproxen produced the linear relationship. However, acemetacin, diclofenac Na, flufenamic acid, indometacin, niflumic acid, tolmetin Na and sulindac presented non linear competition plots manifesting at relatively low drug concentrations. This effect was corrected by increasing DR concentrations from 2.8 mM to 15 mM. The modification did not affect rate constants values for those derivatives which already presented a linear plot at 2.8 mM, but allowed to calculate rate constants for other compounds. It is suggested that the experimental conditions have to be adapted particularly for those derivatives with a relatively high. rate constant for reaction with the radical species. The oxicam derivatives (tenoxicam and piroxicam) presented another kind of deviation that revealed a prooxidant effect in this system: non linear plots were also observed at relatively low drug concentrations, but in the opposite direction than for the other molecules. This last effect was independent of DR concentration but could be corrected by increasing ascorbate concentration in the system.     

Continous monitoring of ATP-converting reactions by purified firefly luciferase.

The time course of the bioluminescence obtained with a partially purified firefly luciferase preparation has been studied. At ATP levels less than 10^?6m the light emission could be maintained essentially constant for several minutes, if the luciferase was not subjected to product inhibition or other inactivating processes. This could be achieved by performing the reaction at appropriate pH and concentration of luciferin and luciferase. Under these conditions continuous measurement of light emission may be used for nondestructive monitoring of ATP-converting reactions, since the emission will be proportional to the ATP concentration in each instant. The continuous monitoring of ATP concentration by firefly luciferase was used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. It was found to be extremely sensitive and convenlent for routine applications.