Bioluminescence Reaction Research Articles

Detection of Hispidin by a Luminescent System from Basidiomycete Armillaria borealis.

In in vitro experiments, the possibility of using a luminescent system extracted from the luminous fungus Armillaria borealis has been shown to detect and determine the concentration of hispidin. A linear dependence of the luminescent response on the content of hispidin in solutions in the concentration range of 5.4 × 10-5-1.4 × 10-2 µM was detected. The stability of the enzyme system and the high sensitivity of the bioluminescent reaction allows carrying out multiple measurements with the analyte detection limit of 1.3 × 10-11 g. The obtained results show the prospects of creating a rapid bioluminescent method for the analysis of medical substances or extracts from various biological objects for the presence of hispidin.

A swinging seesaw as a novel model mechanism for time-dependent hormesis under dose-dependent stimulatory and inhibitory effects: A case study on the toxicity of antibacterial chemicals to Aliivibrio fischeri

Hormesis occurs frequently in broadly ranging biological areas (e.g. plant biology, microbiology, biogerontology), toxicology, pharmacology and medicine. While numerous mechanisms (e.g. receptor and pathway mediated pathway responses) account for stimulatory and inhibitory features of hormetic dose responses, the vast majority emphasizes the inclusion of many doses but only one timepoint or use of a single optimized dose that is assessed over a broad range of timepoints. In this paper, a toxicity study was designed using a large number of properly spaced doses with responses determined over a large number of timepoints, which could help us reveal the underlying mechanism of hormesis. We present the results of a dose-time-response study on hormesis using five antibacterial chemicals on the bioluminescence of Aliivibrio fischeri, measuring expression of protein mRNA based on quorum sensing, simulating bioluminescent reaction and analyzing toxic actions of test chemicals. The findings show dose-time-dependent responses conforming to the hormetic dose-response model, while revealing unique response dynamics between agent induced stimulatory and inhibitory effects within bacterial growth phase dynamics. These dynamic dose-time features reveal a type of biological seesaw model that integrates stimulatory and inhibitory responses within unique growth phase, dose and time features, which has faultlessly explained the time-dependent hormetic phenomenon induced by five antibacterial chemicals (characterized by low-dose stimulation and high-dose inhibition). This study offers advances in understanding cellular dynamics, the biological integration of diverse and opposing responses and their role in evolutionary adaptive strategies to chemicals, which can provide new insight into the mechanistic investigation of hormesis.

Study of coelenterazine luminescence: Electrostatic interactions as the controlling factor for efficient chemiexcitation

Coelenterazine is a common substrate used by marine species in enzyme-catalyzed bioluminescent reactions, in which thermal energy is converted into excitation energy. Coelenterazine is also known to emit chemiluminescence, in the absence of enzymes. Moreover, the scaffold of this molecule is present in organisms of eight phyla, making it a prototypical system for marine chemi-/bioluminescence. The characterization of the chemiexcitation step responsible for light emission is essential for future applications in bioimaging, bioanalysis and biomedicine. We have found evidence to support the identification of a neutral dioxetanone intermediate as the responsible for efficient chemiexcitation. This is explained by attractive electrostatic interactions between the CO2 and Coelenteramide moieties, which allow the reacting dioxetanone to spend time in a PES region of degeneracy between singlet ground and excited states. Contrary to expected, there is no relationship between electron (ET)/charge (CT) transfer, from an electron-rich moiety to the peroxide, and efficient chemiexcitation. Thus, neither Chemically Induced Electron-Exchange Luminescence (CIEEL) nor Charge Transfer-Initiated Luminescence (CTIL) can be used to explain imidazopyrazinone-based chemi-/bioluminescence. We have also found a concentration-dependent quenching effect, more prevalent at acidic pH.

Mechanistic explanation of time-dependent cross-phenomenon based on quorum sensing: A case study of the mixture of sulfonamide and quorum sensing inhibitor to bioluminescence of Aliivibrio fischeri

Cross-phenomenon in which the concentration-response curve (CRC) for a mixture crosses the CRC for the reference model has been identified in many studies, expressed as a heterogeneous pattern of joint toxic action. However, a mechanistic explanation of the cross-phenomenon has thus far been extremely insufficient. In this study, a time-dependent cross-phenomenon was observed, in which the cross-concentration range between the CRC for the mixture of sulfamethoxypyridazine (SMP) and (Z-)-4-Bromo-5-(bromomethylene)-2(5H)-furanone (C30) to the bioluminescence of Aliivibrio fischeri (A. fischeri) and the CRC for independent action model with 95% confidence bands varied from low-concentration to higher-concentration regions in a timely manner expressed the joint toxic action of the mixture changing with an increase of both concentration and time. Through investigating the time-dependent hormetic effects of SMP and C30 (by measuring the expression of protein mRNA, simulating the bioluminescent reaction and analyzing the toxic action), the underlying mechanism was as follows: SMP and C30 acted on the quorum sensing (QS) system of A. fischeri, which induced low-concentration stimulatory effects and high-concentration inhibitory effects; in the low-concentration region, the stimulatory effects of SMP and C30 made the mixture produce a synergistic stimulation on the bioluminescence; thus, the joint toxic action exhibited antagonism. In the high-concentration region, the inhibitory effects of SMP and C30 in the mixture caused a double block in the loop circuit of the QS system; thus, the joint toxic action exhibited synergism. With the increase of time, these stimulatory and inhibitory effects of SMP and C30 were changed by the variation of the QS system at different growth phases, resulting in the time-dependent cross-phenomenon. This study proposes an induced mechanism for time-dependent cross-phenomenon based on QS, which may provide new insight into the mechanistic investigation of time-dependent cross-phenomenon, benefitting the environmental risk assessment of mixtures.

Lessons Learned from Luminous Luciferins and Latent Luciferases.

Compared to the broad palette of fluorescent molecules, there are relatively few structures that are competent to support bioluminescence. Here, we focus on recent advances in the development of luminogenic substrates for firefly luciferase. The scope of this light-emitting chemistry has been found to extend well beyond the natural substrate and to include enzymes incapable of luciferase activity with d-luciferin. The broadening range of luciferin analogues and evolving insight into the bioluminescent reaction offer new opportunities for the construction of powerful optical reporters of use in live cells and animals.

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction.

We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions. Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes. A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.

Analytical Enzymatic Reactions in Microfluidic Chips

A number of approaches have been proposed and tested to transfer enzymatic reactions into the functional elements of microfluidic chips on the example of the bienzyme bioluminescent reaction involving NAD(P)H:FMN-oxidoreductase and luciferase. Measurement of the catalytic activity of these enzymes (under the influence of pollutants) is the basis of enzymatic bioassay of various liquids. It was found that all of the components of the reaction must be placed in the same cell of the chip to improve the reproducibility of the measurements. The use of starch gel as a carrier for immobilization and gelatin as a scaffold in the reactor of the chip enables the preservation of enzyme activity in the course of sealing the chip at room temperature. It is shown that the components of the reaction should be vigorously stirred in a microfluidic chip reactor to improve the efficiency of the analysis. As a result of the studies, a prototype of microfluidic chip based on the enzymatic bioluminescent reaction is proposed. It is characterized by a detection limit of copper sulfate of 3 μM that corresponds to the sensitivity of traditional lux-biosensors based on living cells. The analysis time is reduced to 1 min, and the analysis can be performed by individuals without special laboratory skills.

Constant pH Accelerated Molecular Dynamics Investigation of the pH Regulation Mechanism of Dinoflagellate Luciferase.

The bioluminescence reaction in dinoflagellates involves the oxidation of an open-chain tetrapyrrole by the enzyme dinoflagellate luciferase (LCF). The activity of LCF is tightly regulated by pH, where the enzyme is essentially inactive at pH ∼8 and optimally active at pH ∼6. Little is known about the mechanism of LCF or the structure of the active form of the enzyme, although it has been proposed that several intramolecularly conserved histidine residues in the N-terminal region are important for the pH regulation mechanism. Here, constant pH accelerated molecular dynamics was employed to gain insight into the conformational activation of LCF induced by acidification.

Quantum yield improvement of red-light-emitting firefly luciferin analogues for in vivo bioluminescence imaging

The dimethylamino group of AkaLumine ((4S)-2-[(1E,3E)-4-[4-(dimethylamino)phenyl]-1,3-butadien-1-yl]-4,5-dihydro-4-thiazolecarboxylic acid), a red-light-emitting firefly luciferin analogue, was replaced by cyclic amino groups (1-pyrrolidinyl, 1-piperidino, 1-azepanyl, and 4-morpholino) to give AkaLumine analogues exhibiting desirable bioluminescence with emission maxima in the red region (656–667 nm). In particular, a bioluminescence reaction of 1-pyrrolidinyl analogue with a recombinant Photinus pyralis luciferase showed a higher quantum yield than that with AkaLumine, giving an improved bioluminescence intensity. The 1-pyrrolidinyl analogue also showed the strongest luminescence in whole-body luciferase-expressing mice among the analogues, indicating that a quantum yield improvement of a luciferin analogue is effective to increase bioluminescence imaging intensity.

Structure of fungal oxyluciferin, the product of the bioluminescence reaction.

The structure of fungal oxyluciferin was determined, the enzymatic bioluminescence reaction under substrate saturation conditions with discrete monitoring of formed products was conducted, and the structures of the end products of the reaction were established. On the basis of these studies, the scheme of oxyluciferin degradation to the end products was developed. The structure of fungal oxyluciferin was confirmed by counter synthesis.

Combined experimental and theoretical study of Coelenterazine chemiluminescence in aqueous solution

Coelenterazine is a common substrate used by marine species in enzyme-catalyzed bioluminescent reactions, in which thermal energy is converted into light-emission. Besides bioluminescence, Coelenterazine is also known to emit chemiluminescence in aprotic solvents. We report here the study of Coelenterazine chemiluminescence in aqueous solution. Water inhibits light-emission even in mixtures with water content as low as 20%. Moreover, we provide convincing spectroscopic evidence that the presence of water affects the ground state (S0) chemical reaction, and not the excited state processes (as chemiexcitation and the fluorescent quantum yield). However, the energetics of the S0 chemical reaction is not affect by addition of water, which points to the inhibition being caused by the reduced lifetime of superoxide anion in water, which is an intermediate in the luminescent reactions of Coelenterazine. This finding indicates that one of the catalytic roles of bioluminescent enzymes is to extend the lifetime of this radical.

Mechanistic Insight into Cypridina Bioluminescence with a Combined Experimental and Theoretical Chemiluminescent Approach.

The bioluminescent reaction of the "sea firefly" Cypridina hilgendorfii is a prototypical system for marine bioluminescence, as its substrate possesses an imidazopyrazinone core that is a common link among organisms of eight phyla. The elucidation of the mechanism behind Cypridina bioluminescence is essential for future applications in bioimaging, biomedicine, and bioanalysis. In this study we have investigated the key step of chemiexcitation with a combined experimental and theoretical approach. The obtained results indicate that neutral dioxetanone is responsible for efficient chemiexcitation, as the thermolysis of this species gives access to a long region of the potential energy surface (PES), where the ground and excited singlet states are degenerated. Contrary to expected, neither chemically induced electron-exchange luminescence (CIEEL) nor charge transfer-initiated luminescence (CTIL) can be used to explain imidazopyrazinone-based bioluminescence, as there is no clear relationship between electron (ET)/charge (CT) transfer (occurring between the electron-rich moiety and dioxetanone) and chemiexcitation. Attractive electrostatic interactions between the CO2 and oxyluciferin moieties allow neutral dioxetanone to spend time in the PES region of degeneracy, while repulsive interactions for anionic dioxetanone lead to a quicker CO2 detachment.

Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase

BackgroundLuciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.MethodsHere, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.ResultsIn this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method.ConclusionWith this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.

Four‐membered cyclic peroxides: Carriers of chemical energy

AbstractFour‐membered cyclic peroxides are high‐energy compounds often associated to cold light emission, but whose chemical and biological roles are still matters of debate. The often‐dangerous synthesis of 1,2‐dioxetanes, achieved around 50 years ago, has been mastered over the years to a point where some derivatives are commercially available. This fact does not imply that 1,2‐dioxetanes can be conveniently prepared in the gram scale or that the synthesis of analogous 1,2‐dioxetanones and the elusive 1,2‐dioxetanedione are simple. Important questions on the mechanism of chemiluminescence and bioluminescence reactions are under experimental and theoretical scrutiny. The available data have contributed to relate structural and medium effects to the quantum efficiency of these compounds to produce excited states. Consequently, such peroxides have been suggested to produce biologically relevant electronically excited species in vivo in the absence of light. The connection of this hypothesis with melanin‐mediated photodamage in the dark has renewed the interest in such cyclic peroxides. This review gives some insight on the synthesis, chemiluminescence mechanism, and biological relevance of 1,2‐dioxetanes, 1,2‐dioxetanones, and 1,2‐dioxetanedione and provides practical protocols for those interested in engaging this field.

Photoinactivation related dynamics of ctenophore photoproteins: Insights from molecular dynamics simulation under electric-field

Photoinactivation is a common phenomenon in bioluminescence ctenophore photoproteins (e.g mnemiopsin, berovin and BfosPP) with still unknown mechanism. The activity of coelenterate photoproteins (e.g aequorin), which has high structural similarity with ctenophore photoproteins, is not affected by light. Recently, we have characterized the effects of light on ctenophore photoprotein mnemiopsin, in different conformations, which has demonstrated light induced structural changes, uniquely secondary structures, of both apo and holo mnemiopsin. This paper is further expansion of our previous work, by applying molecular dynamics simulations to investigate photoinactivation related dynamics of berovin at atomistic level, in comparison with aequorin, under the influence of electric component of electromagnetic field. The results have indicated that the intense electric filed could influence structure of both berovin and aequorin but in different manner, whereas moderate electric field only effects on berovin's structure remarkably. In this case, increased helicity of residues E180-M193 and decreased helical contents of L38-D46 and L125-D138 segments are considerable in berovin as well as flexibility elevation of calcium binding loops. These changes cause structural expansion of berovin, especially at N-terminal domain, in direction of electric field. In conclusion, the induced structural changes of mentioned helical parts together with elevated fluctuation of their adjacent segments, N26-D46 and M193-Y206, indicate the influence of light on substrate stabilizing residues, Arg41 and Y204. This condition could presumably leads to inactivation of bioluminescence reaction due to separation of substrate from the cavity of the protein.

Active mixing of immobilised enzymatic system in microfluidic chip

Parameters for sample introduction, dried reagents dissolution and mixing with sample for bienzyme system NAD(H):FMN-oxidoreductase and luciferase immobilised in microfluidic chip were successfully determined. Numerical simulations of reaction chamber geometry, flavin mononucleotide (FMN) escape from starch gel and mixing options were conducted to achieve higher sensitivity of bioluminescent reaction. Results of numerical simulations were verified experimentally. The active mixer for dried reagents was made from an electro-mechanical speaker's membrane which was connected to the input of the chip. Such a mixer provided better efficiency than a passive mixing, and it is simple enough for use in point-of-care devices with any systems based on immobilised enzymes in chips.

Fluorescent coelenteramide-containing protein as a color bioindicator for low-dose radiation effects

The study addresses the application of fluorescent coelenteramide-containing proteins as color bioindicators for radiotoxicity evaluation. Biological effects of chronic low-dose radiation are under investigation. Tritiated water (200MBq/L) was used as a model source of low-intensive ionizing radiation of beta type. 'Discharged obelin,' product of bioluminescent reaction of marine coelenterate Obelia longissimi, was used as a representative of the coelenteramide-containing proteins. Coelenteramide, fluorophore of discharged obelin, is a photochemically active molecule; it produces fluorescence forms of different color. Contributions of 'violet' and 'blue-green' forms to the visible fluorescence serve as tested parameters. The contributions depend on the coelenteramide's microenvironment in the protein, and, hence, evaluate distractive ability and toxicity of radiation. The protein samples were exposed to beta radiation for 18days, and maximal dose accumulated by the samples was 0.28Gy, being close to a tentative limit of a low-dose interval. Increase of relative contribution of 'violet' fluorescence under exposure to the beta irradiation was revealed. High sensitivity of the protein-based test system to low-dose ionizing radiation (to 0.03Gy) was demonstrated. The study develops physicochemical understanding of radiotoxic effects. Graphical abstract Coelenteramide-containing protein (discharged obelin) changes fluorescence color under exposure to low-dose ionizing radiation of tritium.

Light emission miracle in the sea and preeminent applications of bioluminescence in recent new biotechnology

Bioluminescence is referred to the light emission by a living organism due to a specific biochemical reaction. This interesting feature of the organisms could highly influences behavioral and ecosystem dynamics. Luminescence, mostly observed in marine species, is generally higher in deep-living genera than in benthic or shallow organisms. However, among creatures living in land, fireflies, beetles, springtails and fungi have shown some bioluminescent activities. Classically, the emission of light is catalyzed by luciferase from a substrate. Interestingly, light-emitting organisms are more abundant and widespread in marine than terrestrial environments. Novel tools derived from understanding bioluminescent reactions have led to countless valuable applications in modern biotechnology and biochemical engineering. Here, we overview some main properties bioluminescence in marine organism from bacteria to fishes following the latest advances and new discoveries of state-of-the-art bioluminescent tools in molecular biology, bioluminescent bioassays and imaging. The overview showed available and wide biotechnological tools of bioluminescence take advantage of its high detectability, high sensitive, low toxic and quantum efficiency which make wide usage as reporter of many biological functions in different fields, such as studying bacterial pathogens, ecotoxicology, food toxicity, tracking cells of interest in vivo, protein–protein interactions, gene expression and circadian rhythms. With the recent invention of luminescent reporters, future possibilities for the development of additional reporter applications are promising.

Inhibition of bioluminescence in the living gills of the luminous fungus Mycena chlorophos by trans-4-aminocinnamic acid

The living gills of the fungus Mycena chlorophos spontaneously emit green light. It was previously reported that trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnnamic acid are essential for the bright light production in the living gills. However, the chemical mechanisms underlying their bioluminescence are unknown. In the present study, trans-4-aminocinnamic acid was found to inhibit light production in the living gills. The concentrations of trans-4-aminocinnamic acid that inhibited the bioluminescence intensity by 50% of initial values for immature and mature gills were 0.07 μM and 4 μM, respectively. Approximately 20% of the bioluminescence intensity of the immature and mature gills was not inhibited by a further increase in the concentration of trans-4-aminocinnamic acid. Moreover, the bioluminescence that was activated by trans-4-hydroxycinnamic acid or trans-3,4-dihydroxycinnamic acid (0.01 mM) was completely inhibited by trans-4-aminocinnamic acid. Therefore, trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid functioned for the bioluminescence that was inhibited by trans-4-aminocinnamic acid. trans-4-Aminocinnamic acid strongly bound to the bioluminescence system(s) and withstood rinsing of the gills with 10 mM phosphate buffer (pH = 7), and high concentrations of trans-4-hydroxycinnamic acid (1 mM) and trans-3,4-dihydroxycinnamic acid (0.1 mM) functioned to displace trans-4-aminocinnamic acid from the bioluminescence system(s) and reactivate bioluminescence. Benzenamine, trans-cinnamic acid, trans-2-aminocinnamic acid, and trans-3-aminocinnamic acid did not inhibit bioluminescence. Therefore, the structure-specific inhibition by trans-4-aminocinnamic acid suggested that the 4-hydroxy group in trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid molecules plays a functional role in the bioluminescence reaction.

Double-Enzymes-Mediated Bioluminescent Sensor for Quantitative and Ultrasensitive Point-of-Care Testing.

We report an ultrasensitive, quantitative, and rapid bioluminescent immunosensor (ABS) for point-of-care testing (POCT) of the disease biomarker in clinical samples using double enzymes including alkaline phosphatase (ALP) and luciferase. In the presence of the biomarker, the ALP attached on the surface of immuno-nanocomplex dephosphorylates adenine triphosphate (ATP), subsequently inhibiting the ATP-luciferin-luciferase bioluminescent reaction. The highly sensitive response of ATP (picomolar level) allows for ultrasensitive detection of biomarker via the effective change of the bioluminescence intensity through ALP- and luciferase-catalyzed reactions, which can be quantitatively determined by a portable ATP detector. This ABS fulfills the criteria for POCT that performs sensitive (femtomolar level of biomarkers) and quantitative measurement quickly (less than 1 h) with minimal equipment (portable detector).