Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).     

The roles of the two highly unstable components F and P involved in the bioluminescence of euphausiid shrimps

Bioluminescence of euphausiids takes place when a fluorescent tetrapyrrole F and a highly unstable protein P react in the presence of oxygen. A previous study on the euphausiid Meganyctiphanes norvegica indicated that F acts as a catalyst and P is consumed in the luminescence reaction, differing from the luminescence system of dinoflagellates in which a tetrapyrrole luciferin, nearly identical to F, is enzymatically oxidized in the presence of dinoflagellate luciferase. In the present study, P was extracted from Euphausia pacifica as well as from M. norvegica, then purified separately by affinity chromatography on a column of biliverdin–Sepharose 4B, completing the whole process in less than 5h. The samples of P obtained from both species had a molecular weight of 600,000, a purity of about 80%, and a specific activity 50–100 times greater than that previously found. The activity of P rapidly decreased in solutions, even at 0°C, and the inactivation of P derived from M. norvegica was more than four times faster than that derived from E. pacifica. The kinetics of the luminescence reaction was investigated with F and P whose concentrations were systematically varied. The reaction was characteristically slow and involved two different reaction rates; the turnover number at 0°C was 30/h for the initial 20 min and 20/h after the initial 1 h. The total light emitted in a 50-h period indicated that the bioluminescence quantum yield of F was about 0.6 at 0°C, and P recycled many times in the luminescence reaction. Thus, the present results conclusively show that F is a luciferin and P is a luciferase of an unusually slow-working type, contrary to early report.     

Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.     

Protein structure and bioluminescent spectra for firefly bioluminescence

Modern theory on general and specific effects of microenvironment on emission spectra was used for explanation of spectral differences for both natural and mutant forms of beetle luciferases, as well as for bioluminescence emitter oxyluciferin in model systems. For the analysis, both authors' and other published data were used. It was shown that active site mutations that resulted in spectral shifts of bioluminescence as a rule caused substantial decrease in the catalytic activity of the enzyme. At the same time, mutations in the conservative regions of the protein amino acid sequence that were in the periphery of the protein globe resulted in red shift of the bioluminescence spectra without affecting catalytic activity. Correlation was observed between the value of spectral shift and polarizability of the introduced amino acid residue: the higher the polarizability, the larger was the red shift of bioluminescence. Copyright © 2002 John Wiley & Sons, Ltd.     

The expression level of luciferase within tumour cells can alter tumour growth upon in vivo bioluminescence imaging.

In vivo bioluminescence imaging is becoming a very important tool for the study of a variety of cellular and molecular events or disease processes in living systems. In vivo bioluminescence imaging is based on the detection of light emitted from within an animal. The light is generated as a product of the luciferase-luciferin reaction taking place in a cell. In this study, we implanted mice with tumour cells expressing either a high or a low level of luciferase. In vivo bioluminescence imaging was used to follow tumour progression. Repeated luciferin injection and imaging of high and low luciferase-expressing tumours was performed. While low luciferase-expressing tumours grew similarly to vector controls, growth of the high luciferase-expressing tumours was severely inhibited. The observation that a high level of luciferase expression will inhibit tumour cell growth when an animal is subjected to serial in vivo bioluminescence imaging is potentially an important factor in designing these types of studies.     

Stability of d‐luciferin for bioluminescence to detect gene expression in freely moving mice for long durations

Circadian disturbance of clock gene expression is a risk factor for diseases such as obesity, cancer, and sleep disorders. To study these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate d ‐luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little information is known about the stability of d ‐luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of a luciferin solution over 21 days. l ‐Luciferin, which is synthesized using racemization of d ‐luciferin, was at high concentrations after 21 days. In addition, we showed that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased compared with the day 1 solution, although locomotor activity rhythm was not affected. These results showed that d ‐luciferin should be applied to the mouse within, at most, 7 days to detect bioluminescence of Per1 gene expression rhythm in vivo.     

Properties and cellular localization of a luciferin binding protein in the bioluminescence reaction of Gonyaulax polyedra

A luciferin binding protein LBP involved in the bioluminescence reaction of Gonyaulax polyedra was purified and used for antibody production. Luciferin bound to LBP is fluorescent and can be used as a marker in living cells, allowing the localization of LBP in cortical organelles to be visualized. In cell sections, the same peripheral localization was observed using anti-LBP and immunofluorescence microscopy. The amount of LBP is ten-fold greater from cells from in night phase compared to those from in day phase, as determined both by immunoblots of cell extracts, and in vivo fluorescence. These changes correlate with the circadian changes in bioluminescence of living cells.     

A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase

Prostaglandin E₂ is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competitive immunosorbent assay for prostaglandin E₂ utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E₂ amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandin E₂ required to displace 50% of the maximal binding of Cypridina luciferase‐labeled prostaglandin E₂ (B/B₀) was approximately 35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme‐linked immunosorbent assay. Copyright © 2009 John Wiley & Sons, Ltd.     

ATP生物发光测定试剂研究进展

萤火虫荧光素酶是ATP生物发光试剂的关键组成部分,可通过萤火虫尾提取纯化或基因工程技术制备,酶的活力和纯度决定了ATP生物发光试剂的性能。迄今许多先进技术在ATP生物发光试剂的制备中均有应用,包括酶基因工程改造技术、ATP循环的酶法放大技术、荧光素酶蛋白的活力及发光稳定技术,特异的细胞ATP提取技术等。ATP生物发光试剂的研究焦点主要集中在提高发光试剂的检测灵敏度和性能、增加产品的适应性等方面。     

Comparison of the thermostability of recombinant luciferases from Brazilian bioluminescent beetles: Relationship with kinetics and bioluminescence colours

Firefly luciferases have been used extensively as bioanalytical reagents and their cDNAs as reporter genes for biosensors and bioimaging, but they are in general unstable at temperatures above 30°C. In the past few years, efforts have been made to stabilize some firefly luciferases for better application as analytical reagents. Novel luciferases from different beetle families, displaying distinct bioluminescence colours and kinetics, may offer desirable alternatives to extend the range of applications. In the past years, our group has cloned the largest variety of luciferases from the three main families of bioluminescent beetles (Elateridae: P. termitilluminans, F. bruchi, P. angustus; Phengodidae: P. hirtus, P. vivianii; and Lampyridae: A. vivianii, C. distinctus and Macrolampis sp2) occurring in Brazilian biomes. We compared the thermostability of these recombinant luciferases and investigated their relationships with bioluminescence spectra and kinetics. The most thermostable luciferases were those of Pyrearinus termitilluminans larval click beetle (534 nm), Amydetes vivianii firefly (539 nm) and Phrixotrix vivianii railroad worm (546 nm), which are the most blue‐shifted examples in each family, confirming the trend that the most blue‐shifted emitting luciferases are also the most thermostable. Comparatively, commercial P. pyralis firefly luciferase was less thermostable than P. termitilluminans click beetle and A. vivianii firefly luciferases. The higher thermostability in these luciferases could be related to higher degree of hydrophobic packing and disulfide bond content (for firefly luciferases).     

Fire fly luciferin as antioxidant and light emitter: the evolution of insect bioluminescence.

Insects are the main group with luminescent species among terrestrial animals. In this paper, we report that fire fly luciferin is endowed with antioxidant properties against oxidative and nitrosative stress. The luciferin reduces linoleate peroxidation in acellular tests and increases the viability of mammalian cells exposed to the oxidant tert-butyl hydroperoxide. Dehydrorhodamine-based tests indicate that fire fly luciferin also scavenges peroxynitrite, whereas parallel tests on cells showed a marked protection of cells subjected to the peroxynitrite generator SIN-1. Together, these results suggest that fire fly luciferin's antioxidant properties could help photocytes coping with the hyperoxidant conditions to which they are submitted during luminous emissions. These data could also suggest that the evolutionary foundation of the bioluminescent system could have been the luciferin, and not the luciferase, first serving as a scavenger of oxidants toxic to the cells, then as a light emitting substrate for luciferase precursors. Similarities with the evolutionary scenario proposed for marine bioluminescent organisms relying on coelenterazine suggest that the surprisingly high success rate observed in the independent emergence of bioluminescent animals could reflect the ease of transformation of antioxidant mechanisms into light-producing systems.     

Extended‐release PEG–luciferin allows for long‐term imaging of firefly luciferase activity in vivo

Bioluminescence has gained favour in the last decade as an approach for observing tumours in vivo in a non‐destructive manner. This very sensitive technique is based on light emission by the reaction of luciferin with the enzyme luciferase, as measured by a photodetector. Ever since the development of recombinant tumour cell lines that have been engineered to produce luciferase, a vast number of experiments have been carried out examining tumour growth, tumour metastasis and the effect of therapeutic regimens in such cases. A primary stumbling block, however, is the relatively short circulatory half‐life of luciferin. In this paper, we propose the PEGylation of 6‐amino‐d ‐luciferin to extend its in vivo circulatory half‐life, thus making the possibility of long‐term observations in animals possible. The covalent attachment was through a carbamate linker that is known to hydrolyse in vivo, releasing the parent compound. Based on our studies, longer emission of the PEGylated luciferin was observed, as compared to free luciferin in mice bearing PC3 prostate tumours expressing luciferase. This result suggests that this reagent can be used in applications requiring extended monitoring of luciferase activation in vivo. Copyright © 2008 John Wiley & Sons, Ltd.     

Copper-mediated oxidation of imidazopyrazinones inhibits marine luciferase activity

The development of bioluminescence-based tools has seen steady growth in the field of chemical biology over the past few decades ranging in uses from reporter genes to assay development and targeted imaging. More recently, coelenterazine-utilizing luciferases such as Gaussia, Renilla, and the engineered nano-luciferases have been utilized due to their intense luminescence relative to firefly luciferin/luciferase. The emerging importance of these systems warrants investigations into the components that affect their light production. Previous work has reported that one marine luciferase, Gaussia, is potently inhibited by copper salt. The mechanism for inhibition was not elucidated but was hypothesized to occur via binding to the enzyme. In this study, we provide the first report of a group of nonhomologous marine luciferases also exhibiting marked decreases in light emission in the presence of copper (II). We investigate the mechanism of action behind this inhibition and demonstrate that the observed copper inhibition does not stem from a luciferase interaction but rather the chemical oxidation of imidazopyrazinone luciferins generating inert, dehydrated luciferins.     

Luciferin derivatives in bioluminescence-enhanced enzyme immunoassays

Ultrasensitive bioluminescence immunoassays for the determination of peptides and proteins (illustrated with human urinary kallikrein, bradykinin and the determination of human urinary kallikrein antibody titres) have been developed. The usable ranges of the standard curves are from 5 pg to 5000 pg per litre. The relative intra-assay coefficients of variation of the tests were between 2% and 6%, and the inter-assay coefficients of variation between 4% and 12%.     

Practical application of bioluminescence enzyme immunoassay using enhancer for firefly luciferin–luciferase bioluminescence

Firefly luciferin–luciferase bioluminescence is known for its high quantum yield (41.0 ± 7.4%). Given this high quantum yield, application of this bioluminescence is expected to be useful in the field of clinical diagnostics. The kinetic profile of this bioluminescence exhibits an instant rise (<1 s) and a rapid decay in light emission (decreased to 42% after 5 s). In this study, we applied four enhancers including coenzyme A, inosine5′‐triphosphate sodium salt, sodium tripolyphosphate and potassium pyrophosphate to prolong light emission. When these enhancers were used, luminescence was only decreased to 89, 83, 87 and 82% after 5 s, respectively. These materials modified the kinetic profile of bioluminescence so that the luminescence is more suitable for clinical application. It becomes more suitable because they enable highly sensitive integration and simplification of a device by separating luminescence measurements from dispensing of reagents. Using these enhancers, we then developed a bioluminescent enzyme immunoassay (BLEIA) for hepatitis B virus surface antigen (HBsAg) that employed firefly luciferase as a labeling enzyme. We compared the results obtained from the HBsAg BLEIA method with the conventional chemiluminescent enzyme immunoassay method, and found a satisfactory correlation (r = 0.984, n = 118). Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe

We demonstrate a novel rapid direct detection method for immunohistochemistry, using a bioluminescent probe. An anti‐CEA antibody‐fused far‐red bioluminescent protein can monitor the accumulation of this type of probe in tumour tissues. The bimodal spectrum (λ_max = 460 and 675 nm) of this bioluminescent probe is extremely stable under different conditions of pH and ion concentration. The sensitivity of our bioluminescent labelling was at the same level of enzymatic labelling, e.g. peroxidase, as an indirect system. Our novel technique is simple and can shorten the pretreatment time of paraffin sections to around 30 min. The utility of our bioluminescent labelling covers all imaging in vitro, in vivo and ex vivo, suggesting that our antibody‐fused bioluminescent probe has the potential to detect tumour antigens with a high sensitivity in routine immune histological examinations. Copyright © 2012 John Wiley & Sons, Ltd.     

活体动物体内光学成像技术的研究进展

生物发光和荧光成像作为近年来新兴的活体动物体内光学成像技术,以其操作简便及直观性成为研究小动物活体成像的一种理想方法,在生命科学研究中得以不断发展。利用这种成像技术,可以直接实时观察标记的基因及细胞在活体动物体内的活动及反应。利用光学标记的转基因动物模型可以研究疾病的发生发展过程,进行药物研究及筛选等。本文综述了现有活体动物体内光学成像技术的原理、应用领域及发展前景,比较了生物发光与几种荧光技术的不同特点和应用。     

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: insights on the origin of protoluciferase activity and bioluminescence colours.

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of d-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site.