RESUMO
Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K+. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems.
Assuntos
Luciferases , Medições Luminescentes , Potássio , Potássio/metabolismo , Potássio/química , Animais , Medições Luminescentes/métodos , Camundongos , Luciferases/química , Luciferases/metabolismo , Humanos , Engenharia de Proteínas , Substâncias Luminescentes/química , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismoRESUMO
The design of π-extended conjugation 'V'-shaped red shifted bioluminescent D-luciferin analogues based on a novel benzobisthiazole core is described. The divergent synthetic route allowed access to a range of amine donor substituents through an SN Ar reaction. In spectroscopic studies, the 'V'-shaped luciferins exhibited narrower optical band gaps, more red-shifted absorption and emission spectra than D-luciferin. Their bioluminescence characteristics were recorded against four different luciferases (PpyLuc, FlucRed, CBR2 and PLR3). With native luciferase PpyLuc, the 'V'-shaped luciferins demonstrated more red-shifted emissions than D-luciferin (λbl =561â nm) by 60 to 80â nm. In addition, the benzobisthiazole luciferins showed a wide range of bioluminescence spectra from the visible light region (λbl =500â nm) to the nIR window (>650â nm). The computational results validate the design concept which can be used as a guide for further novel D-luciferin analogues based upon other 'V'-shaped heterocyclic cores.
Assuntos
Luciferina de Vaga-Lumes , Luz , Luciferina de Vaga-Lumes/química , Luciferases/química , Análise Espectral , Medições Luminescentes/métodos , Luciferases de Vaga-LumeRESUMO
Dinoflagellate luciferin bioluminescence is unique since it does not rely on decarboxylation but is poorly understood compared to that of firefly, bacteria, and coelenterata luciferins. Here we computationally investigate possible protonation states, stereoisomers, a chemical mechanism, and the dynamics of the bioluminescence intermediate that is responsible for chemiexcitation. Using semiempirical dynamics, time-dependent density functional theory static calculations, and a correlation diagram, we find that the intermediate's functional group that is likely responsible for chemiexcitation is a 4-member ring, a dioxetanol, that undergoes [2π + 2π] cycloreversion and the biolumiphore is the cleaved structure. The simulated emission spectra and luciferase-dependent absorbance spectra agree with the experimental data, giving support to our proposed mechanism and biolumiphore. We also compute circular dichroism spectra of the intermediate's four stereoisomers to guide future experiments in differentiating them.
Assuntos
Dinoflagellida , Luciferina de Vaga-Lumes , Luciferina de Vaga-Lumes/química , Luciferinas , Estereoisomerismo , Medições LuminescentesRESUMO
A new rationally designed fully rotationally restricted luciferin has been synthesised. This synthetic luciferin, based upon the structure of infraluciferin, has two intramolecular H-bonds to reduce degrees of freedom, an amine group to enhance ICT process, and an alkenyl group to increase π-conjugation. In the spectroscopic measurements and computational calculations, enamine luciferin showed more red-shifted absorption and fluorescence emission than LH2 and iLH2. With PpyWT luciferase enamine luciferin gave bioluminescence at 564 nm which is similar to LH2 at 561 nm. Further investigation by docking studies revealed that the emission wavelength of enamine luciferin might be attributed to the unwanted twisted structure caused by Asp531 within the enzyme. With mutant luciferase FlucRed, the major emission peak was shifted to 606 nm, a distinct shoulder above 700 nm, and 21% of its spectrum located in the nIR range.
Assuntos
Luciferina de Vaga-Lumes , Luciferinas , Simulação de Acoplamento Molecular , Luciferina de Vaga-Lumes/química , Luciferases/química , Medições Luminescentes/métodosRESUMO
Engineered luciferase-luciferin pairs have expanded the number of cellular targets that can be visualized in tandem. While light production relies on selective processing of synthetic luciferins by mutant luciferases, little is known about the origin of selectivity. The development of new and improved pairs requires a better understanding of the structure-function relationship of bioluminescent probes. In this work, we report a biochemical approach to assessing and optimizing two popular bioluminescent pairs: Cashew/d-luc and Pecan/4'-BrLuc. Single mutants derived from Cashew and Pecan revealed key residues for selectivity and thermal stability. Stability was further improved through a rational addition of beneficial residues. In addition to providing increased stability, the known stabilizing mutations surprisingly also improved selectivity. The resultant improved pair of luciferases are >100-fold selective for their respective substrates and highly thermally stable. Collectively, this work highlights the importance of mechanistic insight for improving bioluminescent pairs and provides significantly improved Cashew and Pecan enzymes which should be immediately suitable for multicomponent imaging applications.
Assuntos
Luciferina de Vaga-Lumes , Medições Luminescentes , Luciferina de Vaga-Lumes/química , Medições Luminescentes/métodos , Luciferases/genética , Luciferases/química , Luciferinas , MutaçãoRESUMO
Due to the strict enantioselectivity of firefly luciferase (FLuc), only D-luciferin can be used as a substrate for the bioluminescence (BL) reaction. Unfortunately, luciferin racemizes easily and accumulation of nonluminous L-luciferin has negative influences on the light-emitting reaction. By a detailed analysis of luciferin chirality, however, it becomes clarified that L-luciferin is the biosynthetic precursor of D-luciferin in fireflies and undergoes the enzymatic chiral inversion. By the chiral inversion reaction, the enantiopurity of luciferin can be maintained in the reaction mixture for applications using FLuc. Thus, chirality is crucial for the BL reaction and essential for investigating and applying the biosynthesis of D-luciferin. Here, we describe the methods for the analysis of chiral inversion reaction using high-performance liquid chromatography (HPLC) with a chiral column. We also introduce an example of an in vitro deracemizative BL reaction system using a combination of FLuc and fatty acyl-CoA thioesterase, which is inspired by the chiral inversion mechanism in the biosynthetic pathway of D-luciferin.
Assuntos
Luciferina de Vaga-Lumes , Luciferinas , Animais , Vaga-Lumes , Luciferina de Vaga-Lumes/química , Luciferases/genética , Luciferases/metabolismo , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismoRESUMO
Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH2 and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with luc genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI.
Assuntos
Besouros , Luciferinas , Animais , Luciferina de Vaga-Lumes/química , Células HEK293 , Humanos , Raios Infravermelhos , Luciferases/química , Luciferases/genética , Medições Luminescentes/métodosRESUMO
Among numerous bioluminescent organisms, firefly is the most studied one. Recent experiment proposed that sulfoluciferin (SLH2 ) may serve as a storage form of luciferin (LH2 ). In the present article, we employed density functional theory calculation to uncover the mechanism and detailed process of the storage and release reactions. Due to lack of available crystallographic structure of the related enzyme, the calculation was performed on a model system. For the storage reaction, possible amino acid residues were used for imitating the protein environment. For the release reaction, the dielectric constant of 3.0 was employed to simulate the polarity of the protein cavity. The computational results indicated that the reactions from LH2 to SLH2 and from SLH2 to LH2 are both exergonic, which favor the storage and release processes and coincide with the experimental observation. Basing on experimental and current theoretical study, we supplemented the stages of LH2 storage and release in the entire bioluminescent cycle of firefly. The current theoretical calculation could inspire the study on LH2 storage and release of other bioluminescent organisms.
Assuntos
Vaga-Lumes , Luciferina de Vaga-Lumes , Aminoácidos , Animais , Luciferina de Vaga-Lumes/química , Luciferases de Vaga-Lume/metabolismo , Luciferinas , Medições Luminescentes/métodos , Modelos TeóricosRESUMO
Replacing an N,N-dimethylamino group in a classical fluorophore with a four membered azetidine ring provides an improved luminescence quantum yield. Herein, we extended this strategy to bioluminescent firefly luciferin analogues and evaluated its general validity. For this purpose, four types of luciferin cores were employed, and a total of eight analogues were evaluated. Among these analogues, unexpectedly, only the benzothiazole core analogue benefited from an azetidine substitution and showed enhanced bioluminescence. In addition, fluorescence measurements revealed that an azetidine substitution improved the fluorescence quantum yield by 2.3-times compared to a N,N-dimethylamino group. These findings clarify the differential effects of azetidine substituents in luciferins and present one possible strategy for enhancing photon output in benzothiazole type luciferins through a synthetic approach.
Assuntos
Azetidinas/química , Luciferina de Vaga-Lumes/química , Substâncias Luminescentes/química , Luciferina de Vaga-Lumes/análogos & derivados , Medições Luminescentes , Estrutura MolecularRESUMO
AkaLumine hydrochloride, named TokeOni, is one of the firefly luciferin analogs, and its reaction with firefly luciferase produces near-infrared (NIR) bioluminescence. Prior to studying the bioluminescence mechanism, basic knowledge about the chemical structures, electronic states, and absorption properties of TokeOni at various pH values of solution has to be acquired. In this paper, the absorption spectra for TokeOni and AkaLumine at pH 2-10 were measured. Density functional theory (DFT) calculations, time-dependent DFT calculations, and the vibrational analyses were carried out. The absorption spectra indicate that the chemical forms of TokeOni in solutions are same as those of AkaLumine. The peaks at pH 7-10 in the absorption spectra correspond to the excitation from the ground state of a carboxylate anion of AkaLumine, the peak at pH 2 corresponds to the excitation from the ground state of a carboxylate anion with an N-protonated thiazoline ring and N-protonated dimethylamino group of AkaLumine, and the peak at pH 4 corresponds to the excitation from the ground state of a carboxylate anion with an N-protonated thiazoline ring of AkaLumine.
Assuntos
Vaga-Lumes , Luciferina de Vaga-Lumes , Animais , Ânions , Luciferina de Vaga-Lumes/química , Concentração de Íons de Hidrogênio , Luciferases de Vaga-Lume/químicaRESUMO
Bioluminescent imaging (BLI) is one of the most powerful and widely used preclinical imaging modalities. However, the current technology relies on the use of transgenic luciferase-expressing cells and animals and therefore can only be applied to a limited number of existing animal models of human disease. Here, we report the development of a "portable bioluminescent" (PBL) technology that overcomes most of the major limitations of traditional BLI. We demonstrate that the PBL method is capable of noninvasive measuring the activity of both extracellular (e.g., dipeptidyl peptidase 4) and intracellular (e.g., cytochrome P450) enzymes in vivo in non-luciferase-expressing mice. Moreover, we successfully utilize PBL technology in dogs and human cadaver, paving the way for the translation of functional BLI to the noninvasive quantification of biological processes in large animals. The PBL methodology can be easily adapted for the noninvasive monitoring of a plethora of diseases across multiple species.
Assuntos
Fenômenos Biológicos , Diagnóstico por Imagem/métodos , Medições Luminescentes/métodos , Modelos Animais , Animais , Animais Geneticamente Modificados , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Cães , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismo , Humanos , Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes/instrumentação , Estrutura Molecular , Reprodutibilidade dos TestesRESUMO
Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents.
Assuntos
Diagnóstico por Imagem , Luciferina de Vaga-Lumes/química , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , AnimaisRESUMO
Bioluminescence is the fascinating natural phenomenon by which living creatures produce light. Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent systems but the luciferin-luciferase pairs of only 11 systems have been characterised to-date, whilst other novel systems are currently under investigation. The different luciferin-luciferase pairs have different light emission wavelengths and hence are suitable for various applications. The last decade or so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is now routinely used for gene assays, the detection of protein-protein interactions, high-throughput screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to control neurons. There has also been an increase in blue-sky research such as the engineering of various light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This review focuses on the past, present, and future applications of bioluminescence. We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist.
Assuntos
Biotecnologia , Luciferina de Vaga-Lumes/química , Luminescência , Medições Luminescentes , Animais , Vaga-Lumes , Luciferina de Vaga-Lumes/metabolismoRESUMO
Bioluminescence imaging with luciferase-luciferin pairs is routinely used to monitor cellular functions. Multiple targets can be visualized in tandem using luciferases that process unique substrates, but only a handful of such orthogonal probes are known. Multiplexed studies require additional robust, light-emitting molecules. In this work, we report new luciferins for orthogonal imaging that comprise disubstituted cores. These probes were found to be bright emitters with various engineered luciferases. The unique patterns of light output also provided insight into enzyme-substrate interactions necessary for productive emission. Screening studies identified mutant luciferases that could preferentially process the disubstituted analogues, enabling orthogonal imaging with existing bioluminescent reporters. Further mutational analyses revealed the origins of substrate selectivity. Collectively, this work provides insights into luciferase-luciferin features relevant to bioluminescence and expands the number of probes for multicomponent tracking.
Assuntos
Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismo , Luciferases/metabolismo , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Células HEK293 , Humanos , Medições Luminescentes , Estrutura MolecularRESUMO
Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.
Assuntos
Luciferina de Vaga-Lumes/química , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Trifosfato de Adenosina/química , Alquilação , Indicadores e Reagentes , Luciferases de Vaga-Lume/química , Especificidade por Substrato , TemperaturaRESUMO
Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.
Assuntos
Envelhecimento/genética , Diagnóstico por Imagem/métodos , Luciferina de Vaga-Lumes/química , Corantes Fluorescentes/química , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Potencial da Membrana Mitocondrial/genética , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Dioxóis/farmacologia , Feminino , Luciferina de Vaga-Lumes/metabolismo , Corantes Fluorescentes/metabolismo , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Neoplasias Mamárias Experimentais/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Nigericina/farmacologia , Compostos de PiridínioRESUMO
Bioluminescence imaging with luciferase-luciferin pairs is commonly used for monitoring biological processes in cells and whole organisms. Traditional bioluminescent probes are limited in scope, though, as they cannot be easily distinguished in biological environments, precluding efforts to visualize multicellular processes. Additionally, many luciferase-luciferin pairs emit light that is poorly tissue penetrant, hindering efforts to visualize targets in deep tissues. To address these issues, we synthesized a set of π-extended luciferins that were predicted to be red-shifted luminophores. The scaffolds were designed to be rotationally labile such that they produced light only when paired with luciferases capable of enforcing planarity. A luciferin comprising an intramolecular "lock" was identified as a viable light-emitting probe. Native luciferases were unable to efficiently process the analog, but a complementary luciferase was identified via Rosetta-guided enzyme design. The unique enzyme-substrate pair is red-shifted compared to well-known bioluminescent tools. The probe set is also orthogonal to other luciferase-luciferin probes and can be used for multicomponent imaging. Four substrate-resolved luciferases were imaged in a single session. Collectively, this work provides the first example of Rosetta-guided design in engineering bioluminescent tools and expands the scope of orthogonal imaging probes.
Assuntos
Luciferina de Vaga-Lumes/química , Luciferases/química , Substâncias Luminescentes/química , Medições Luminescentes , Luciferina de Vaga-Lumes/síntese química , Luciferases/metabolismo , Luminescência , Substâncias Luminescentes/síntese química , Estrutura MolecularRESUMO
The complex of the recombinant fusion protein of apoPholasin and glutathione S-transferase (GST-apoPholasin) with non-fluorescent dehydrocoelenterazine (dCTZ) (GST-apoPholasin/dCTZ complex) shows yellow fluorescence at 539 nm by excitation at 430 nm. The GST-apoPholasin/dCTZ complex with a fluorophore (dCTZ*) has considerably weak luminescence activity, converting slowly to a blue fluorescence protein with the emission peak at 430 nm. The main oxidation products from dCTZ* for blue fluorescence were identified as coelenteramine (CTM) and an unreported pyrazine derivative, 3-benzyl-5-(4-hydroxyphenyl)pyrazin-2(1H)-one (CTO) that was confirmed by chemical synthesis.
Assuntos
Apoproteínas/química , Luciferina de Vaga-Lumes/química , Glutationa Transferase/química , Imidazóis/síntese química , Pirazinas/síntese química , Cor , Fluorescência , OxirreduçãoRESUMO
Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.
Assuntos
Dípteros/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Luciferases/metabolismo , Animais , Cromatografia por Troca Iônica , Dípteros/enzimologia , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/isolamento & purificação , Perfilação da Expressão Gênica , Luciferases/química , Luciferases/isolamento & purificação , Medições Luminescentes , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Espectrometria de FluorescênciaRESUMO
Mechanochemical analogues have recently been established for several enzymatic reactions, but they require periodic interruption of the reaction for sampling, dissolution, and (bio)chemical analysis to monitor their progress. By applying a mechanochemical procedure to induce bioluminescence analogous to that used by the marine ostracod Cypridina (Vargula) hilgendorfii, here we demonstrate that the light emitted by a bioluminescent reaction can be used to directly monitor the progress of a mechanoenzymatic reaction without sampling. Mechanical treatment of Cypridina luciferase with luciferin generates bright blue light which can be readily detected and analyzed spectroscopically. This mechanically assisted bioluminescence proceeds through a mechanism identical to that of bioluminescence in solution, but has higher activation energy due to being diffusion-controlled in the viscous matrix. The results suggest that luciferases could be used as light-emissive reporters of mechanoenzymatic reactions.