Luminescent Reaction Research Articles

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

Green bioluminescence in Renilla species is generated by a approximately 100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein-protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and beta-arrestins. We show that complementation-induced BRET allows detection of the GPCR-beta-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.

Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps

Gaussia luciferase secreted by the copepod Gaussia princeps catalyzes the oxidation of coelenterazine to produce blue light. The primary structure of Gaussia luciferase deduced from the cDNA sequence shows two repeat sequences of 71 amino acid residues, suggesting the luciferase consists of two structural domains. Two domains in Gaussia luciferase were expressed independently in Escherichia coli cells, purified and characterized. We found that both domains have luminescence activity with coelenterazine, and the catalytic properties including luminescence spectrum, optimal pH, substrate specificity and luminescence stimulation by halogen ions (Cl −, Br − and I −) are identical to intact Gaussia luciferase. Thus, Gaussia luciferase has two catalytic domains for the luminescence reaction.

Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris

The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35 kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19kOLase, expressed as inclusion bodies, was solubilized with 6 M urea and purified by urea–nickel chelate affinity chromatography. The yield of 19kOLase was 16 mg from 400 ml of cultured cells. 19kOLase in 6 M urea could be refolded rapidly by dilution with 50 mM Tris–HCl (pH 7.8)–10 mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of organic solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1 μM Cu 2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.

Use of 9,10-diphenylanthracene as a contrast agent in chemiluminescence imaging: The observation of spreading of oxidative degradation in thin polypropylene films

Thin films of polypropylene were doped with a chemiluminescence (CL) activator, 9,10-diphenylanthracene (DPA), and were thermally oxidised in a CL imaging apparatus to determine whether heterogeneous oxidation processes such as spreading of oxidation could be observed. The presence of DPA resulted in significantly more intense CL images compared with undoped polymer, due to the efficient chemically induced electron exchange luminescence reaction between DPA and hydroperoxides. Hence, the CL images from DPA-doped PP were used to locate the position of hydroperoxides in the oxidising polymer. For thermal oxidation at 150 and 140 °C hydroperoxides were observed to form in localised regions of the films, whilst other areas remained hydroperoxide free. As the oxidation time increased the concentration of hydroperoxides in these areas increased and they were observed to spread to the remainder of the polymer. Time-resolved line maps from the images indicated that zones with high concentration of hydroperoxides travel through the polymer during oxidation. Integrals of CL images from the thermal oxidation of DPA-doped polymers indicated that a significant degree of oxidation had occurred by the end of the “induction period” for a conventional CL-intensity oxidation–time profile. This is a likely reason why spreading of oxidation has not previously been observed for undoped PP films.

A Bioluminescent-Based, HTS-Compatible Assay to Monitor G-Protein-Coupled Receptor Modulation of Cellular Cyclic AMP

We have developed a novel assay for monitoring changes in intracellular cyclic AMP (cAMP) concentration with high sensitivity (30 +/- 5 fmol [mean +/- standard error of the mean] of cAMP per well) and reproducibility (Z' of > 0.8). The assay is of format amenable to high throughput screening (HTS) in 96-, 384-, and 1,536-well plates, and as a bioluminescent assay is potentially less prone to interferences originating from fluorescent compounds. Because of its high sensitivity, fewer numbers of cells (1,000 cells per well) in low-volume 384-well plates are required to screen for changes in cAMP concentrations. The assay does not rely on the use of antibodies, and thus it does not suffer from changes in the affinity or quality of the antibodies. The assay is based on the fact that cAMP is a potent activator of cAMP-dependent protein kinase (PKA), and activation of PKA can be monitored by measuring ATP utilization in a kinase reaction. The amount of ATP consumed can be measured using a luciferase/luciferin luminescent reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both the activity of PKA and the intracellular concentration of cAMP is observed. Thus, the functional activity of agents that modulate the activity of Galpha(s) or Galpha(i) forms of G-protein-coupled receptors (GPCRs), which cause change in intracellular cAMP, can be monitored by the change in the activity of PKA and the amount of RLU readout. The assay can be performed in two steps and requires only 30 min after cell lysis for completion. The assay has been successfully used to generate 50% effective concentration (EC(50)) values for forskolin, a known direct activator of cellular adenylate cyclases, and EC(50) values for agonists and 50% inhibitory concentration values for antagonists modulating GPCRs that alter adenylate cyclase activity (Galpha(s) and Galpha(i)). Finally, adherent, suspension, and frozen cells have been successfully used in this assay, thus offering flexibility and convenience for many HTS applications.

Purification and partial spectral characterization of a novel luciferin from the luminous enchytraeid Fridericia heliota

A homogeneous luciferin preparation has been obtained from the luminous soil enchytraeid Fridericia heliota, which has an ATP-dependent luminescent system. A procedure for luciferin purification without losing fractions of active luciferase has been developed. The luciferin specific activity is 4000 times increased; its UV absorption spectrum maximum is 294 nm with a local minimum at 262 nm. The luciferin of the enchytraeid F. heliota is significantly different from firefly luciferin, whose luminescent reaction also requires ATP, and it also appears to have no similarities to other known luciferins.

One-step Bioluminescence ATPase Assay for the Evaluation of Neurotoxic Effects of Metal Ions

Membrane-bound ATPases, such as Na,K-ATPase and nucleotide triphospho-diphosphohydrolase (NTPDase), being one of the first targets of a toxic action are generally considered as good markers for estimating toxicity. A bioluminescence assay was applied for fast and sensitive evaluation of heavy metals effect on the rat brain synaptosomal membrane ATPase activity. The assay consists of ATP-consuming reaction catalyzed by synaptic plasma membrane ATPases coupled to the luminescent firefly luciferase reaction, which consumes residual ATP after the course of ATPase reaction. The bioluminescence ATPase assay was applied to study the effect of heavy and transitional metals (Cu2+, Pb2+, Cd2+, Hg2+) on rat brain ATPase activity after assay optimization. All metals applied inhibited synaptic membrane ATPase activity in a concentration dependent manner. The IC 50 values (Hg2+ < Cu2+ < Cd2+ < Pb2+) obtained with the bioluminescence assay were highly correlated with those obtained by the spectrophotometric method. The fast bioluminescence ATPase assay with small sample and substrate requirements could be adjusted for high-throughput environmental and pharmacological screening.

Imidazole-assisted catalysis of luminescence reaction in blue fluorescent protein from the photoprotein aequorin

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a dissociable complex of Ca 2+-bound apoaequorin and coelenteramide, and is identified as a luciferase that catalyzes the oxidation of coelenterazine by molecular oxygen to emit light. Based on the chemical luminescence of coelenterazine oxidation by an acid–base mechanism, we found that the luminescence activity of BFP-aq was stimulated by imidazole at concentrations of 30–300 mM with coelenterazine and its analogues. The kinetic analyses indicate that imidazole has no effect on the binding affinity of coelenterazine to BFP-aq and may act as a catalytic base, accepting a proton from the –NH– group of coelenterazine and stimulating luminescence activity.

Real-Time Detection of Dopamine Released from a Nerve Model Cell by an Enzyme-Catalyzed Luminescence Method and Its Application to Drug Assessment

A real-time observation of neurotransmitter release from a nerve cell is a useful method for not only neuroscience research, but also assessing of the influence of chemicals, including drugs, on the human nervous system. In this study, a more simple and sensitive method for real-time monitoring of dopamine release from a nerve model cell was developed. Highly sensitive detection of dopamine was performed by using tyramine oxidase for dopamine oxidation, which was followed by a luminol luminescence reaction. This enzyme-catalyzed luminescence method was applied to observe dopamine release from the PC12 cell as a nerve model cell upon stimulation with acetylcholine and an acetylcholine receptor agonist. The results demonstrated that the real-time monitoring of the activation of the PC12 cell was easily performed by this method. This method possessed many advantages, such as high sensitivity, rapid measurement and no pretreatment for cells. It might be applied to drug screening and the assessment of harmful influences of food additives and pesticides on the nerves.

The spatial pattern of bioluminescent flashes in the polychaete Eusyllis blomstrandi (Annelida)

Each of the trunk segments of the polychaete Eusyllis blomstrandi is equipped with paired epidermal luminescent domains. They luminesce upon mechanical or electrical stimulation. Light emission can be rapidly turned on and off, appears intracellular and is highly coordinated among the trunk segments. Luminescent light is typically emitted in series of flashes. Light emission in a flash starts locally in a group of segments and recruits adjacent segments at a rate as fast as ≤1 ms/segment. The collapse of light emission at the end of a flash is almost simultaneous in all of the segments involved. In the intact worm, the luminescent reaction usually involves only a posterior group of segments. Facilitation becomes manifest as the consecutive flashes in a series increase in brightness and duration and recruit additional anterior segments that were not active in earlier flashes. The flash series stops abruptly instead of decreasing asymptotically in brightness. In posterior fragments, all the segments participate in flashing luminescence, indicating the loss of an inhibitory effect exerted by the anterior end in the case of whole animals. Posterior fragments survive and are still capable of luminescence weeks after fragmentation although they do not regenerate a head. Immediately upon fragmentation of the worm, the posterior fragment luminesces continuously for some seconds while the anterior part quickly stops light emission. This suggests a decoy and/or a predator-alerting function of prolonged, strong luminescence by the moribund posterior fragment to the benefit of the survival of the anterior fragment.

Catalytic Properties of Domain-Exchanged Chimeric Proteins between Firefly Luciferase andDrosophilaFatty Acyl-CoA Synthetase CG6178

Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the C-terminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the C-terminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the N-terminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.

Expression, purification and characterization of calcium-triggered luciferin-binding protein of Renilla reniformis

The Ca 2+-triggered luciferin-binding protein of Renilla reniformis (RLBP) is a non-covalent complex of apoprotein (apoRLBP) and coelenterazine (luciferin). The gene encoding apoRLBP with 552 nucleotides has been synthesized by assembly PCR methods with synthetic oligonucleotides, and the histidine-tagged apoRLBP expressed as a soluble form in the periplasmic space of Escherichia coli cells. The apoRLBP was purified by nickel chelate chromatography and the procedure yielded 18.2 mg of recombinant apoRLBP from 80 ml of cultured cells with purity greater than 95%. The purified apoRLBP was converted to RLBP by incubation with coelenterazine in the presence of dithiothreitol and the purity of recombinant RLBP was estimated to be over 95% by comparison with the absorption spectral data of native RLBP. When RLBP mixed with Ca 2+, coelenterazine was dissociated from RLBP and was utilized for the luminescence reaction of Renilla luciferase. Also semi-synthetic RLBPs with h-, e-, and Bis-coelenterazines were prepared and characterized.

Expression, purification and immobilization of firefly luciferase on alkyl-substituted Sepharose 4B

North American firefly, Photinus pyralis, luciferase was produced in Escherichia coli BL 21 by using a pET expression vector. The His-tagged luciferase was purified by metal-ion affinity chromatography. This approach enabled an efficient, one-step purification protocol of a genetically modified luciferase with properties similar to those of the authentic counterpart. Alkyl-substituted Sepharose 4B was used for luciferase immobilization. Matrices were prepared by the glycidyl ether method with different degrees of substitution and alkyl chain length, have been used as a non-ionic matrix for immobilization of firefly luciferase through hydrophobic interaction. Exposure of hydrophobic clusters in the protein molecule was confirmed by fluorescence studies using 8-anilino-1-naphthalene-sulfonate as a hydrophobic reporter probe. Immobilization of firefly luciferase took place with relatively retention of their basic kinetic properties such as K m and recovery of activity. The simplicity of adsorption of luciferase on these matrices together with the activity reusability suggests that the method of immobilization was described provide a useful procedure of bioassays when coupled to the firefly luminescence reaction.

Effect of different salts and detergents on luciferin–luciferase luminescence of the enchytraeid Fridericia heliota

The study addresses the effect produced by different inorganic salts and detergents (SDS, Triton X-100, the Tween series) on the ATP-dependent bioluminescent reaction catalyzed by the luciferase of the new earthworm species Fridericia heliota (Annelida: Clitellata: Oligochaeta: Enchytraeidae). It has been shown that the effect of divalent metal salts on luminescence is determined by the action of cations. Three of them – Mg 2+, Mn 2+ and Ca 2+ – can stimulate luciferase activity at concentrations varying within a wide range, and Mn 2+ can act as a 100%-effective substitute for Mg 2+ in F. heliota luminescence reaction in vitro. The inhibitory effect of monovalent metal salts on luminescence is largely determined by the action of the anion part of the molecule. The effectiveness of the inhibitory effect of anions increases in the following order: Cl - < CO 3 - 2 ∼ SO 3 - 2 ∼ Br - < SO 4 - 2 ∼ PO 4 - 3 < NO 3 - < I - ≪ Cr 2 O 7 - 2 ≪ Fe ( CN ) 6 - 3 . Of the sodium salts, dodecyl sulfate, which is an anionic detergent, produces the strongest inhibitory effect on luciferase. On the contrary, nonionic detergents produce a stimulatory effect on the F. heliota luciferase. The action of the most effective of them – Triton X-100 – is determined by its ability to reduce the actual concentration of lipid inhibitors in the reaction mixture.

Chemiluminescent determination of oxalate based on its enhancing effect on the oxidation of methyl red by dichromate

A flow injection configuration for the chemiluminescence determination of oxalate is proposed. The procedure is based on the catalytic effect of oxalate on the oxidation of methyl red by dichromate. The chromium(III) formed catalyzes the luminescent reaction between luminol and hydrogen peroxide. The proposed method is simple, inexpensive, sensitive and suitable for concentrations of oxalate between 0.24 and 8.11 μg ml −1. The applicability of the method was demonstrated by determining oxalate in vegetables.

Rhythmic gene expression in a purple photosynthetic bacterium, Rhodobacter sphaeroides

Circadian rhythms are known to exist in all groups of eukaryotic organisms as well as oxygenic photosynthetic bacteria, cyanobacteria. However, little information is available regarding the existence of rhythmic behaviors in prokaryotes other than cyanobacteria. Here we report biological rhythms of gene expression in a purple bacterium Rhodobacter sphaeroides by using a luciferase reporter gene system. Self-bioluminescent strains of Rb. sphaeroides were constructed, which produced a bacterial luciferase and its substrate, a long chain fatty aldehyde, to sustain the luminescence reaction. After being subjected to a temperature or light entrainment regime, the reporter strains with the luciferase genes driven by an upstream endogenous promoter expressed self-sustained rhythmicity in the constant free-running period. The rhythms were controlled by oxygen and exhibited a circadian period of 20.5 h under aerobic conditions and an ultradian period of 10.6–12.7 h under anaerobic conditions. The data suggest a novel endogenous oscillation mechanism in purple photosynthetic bacteria. Elucidation of the clock-like behavior in purple bacteria has implications in understanding the origin and evolution of circadian rhythms.

Enzymatic and Genetic Characterization of Firefly Luciferase andDrosophilaCG6178 as a Fatty Acyl-CoA Synthetase

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.

Identity of the Emitter in the Bacterial Luciferase Luminescence Reaction: Binding and Fluorescence Quantum Yield Studies of 5-Decyl-4a-hydroxy-4a,5-dihydroriboflavin-5‘-phosphate as a Model

The excited state of 4a-hydroxy-4a,5-dihydroFMN has been postulated to be the emitter in the bacterial bioluminescence reaction. However, while the bioluminescence quantum yield of the luciferase emitter is about 0.16, chemiluminescence and fluorescence quantum yields of earlier flavin models mimicking the luciferase emitter were no more than 10(-5). To further examine the proposed chemical identity of the luciferase emitter, 5-decyl-4a-hydroxy-4a,5-dihydroFMN was prepared as a new flavin model. Both the wild-type Vibrio harveyi luciferase and a catalytically active alphaC106A mutant formed complexes with the flavin model at a 1:1 molar ratio with K(d) values at 2.4 and 1.2 microM, respectively. This flavin model inhibited the activity of both luciferases, suggesting that it was bound to the enzyme active center. While the free flavin model was itself only very weakly fluorescent, its binding to either luciferase species resulted in markedly enhanced fluorescence, peaking at 440 nm. The fluorescence quantum yields of 5-decyl-4a-hydroxy-4a,5-dihydroFMN bound to wild-type and alphaC106A luciferases were 0.08 and 0.05, respectively, which are about 50% of the respective emitter bioluminescence quantum yields of these two luciferases. The present findings clearly demonstrated that the luciferase active site was suitable for marked enhancement of fluorescence of 4a-hydroxyflavin and, hence, provides a strong support to the proposed identity of 4a-hydroxy-4a,5-dihydroFMN, in its exited state, as the luciferase emitter.

Identification of the biosynthetic units of Cypridina luciferin in Cypridina ( Vargula) hilgendorfii by LC/ESI-TOF-MS

In a luminous ostracod Cypridina ( Vargula) hilgendorfii, Cypridina luciferin with an imidazopyrazinone structure (3,7-dihydroimidazopyrazin-3-one) is utilized for the luminescence reaction. To identify the biosynthetic units of Cypridina luciferin, the stable isotope labeled compounds were examined by feeding experiments with living Cypridina specimens. The incorporation of the labeled compounds into Cypridina luciferin was identified by the method of LC/ESI-TOF-MS analyses and these results suggested that l-tryptophan, l-arginine and l-isoleucine are structural units of Cypridina luciferin.

Effect of red and near-infrared laser light on adenosine triphosphate (ATP) in the luciferine–luciferase reaction

Adenosine triphosphate (ATP) is an important molecule in biology because it stores chemical energy and releases it to the biochemical processes occurring in the cell. In this study we analysed the biochemical behaviour of ATP after irradiating it with 635 and 830nm diode lasers. We analysed the luminescence peak, the reaction rate and the area under the luminescence curve at 2×10−9mol/l of ATP in the luciferine–luciferase luminescence reaction before and after irradiating the molecule at several irradiances and radiant exposures. The absorption spectrum of ATP at 3×10−3mol/l concentration was measured between 650 and 900nm after laser irradiation at 635nm (Argon-Dye) and 830nm (diode laser). We found significant differences in the measured parameters when ATP was irradiated with both wavelengths. The absorption spectra of non-irradiated and irradiated ATP show a physical–chemical difference in the ATP molecule after irradiation with both lasers. We can conclude that visible and near-IR laser light with the parameters that were used in this study changed the biochemical behaviour of ATP molecules.