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.     

New,sensitive, radioactive-free bioluminescence-enhanced detection system in protein blotting and nucleic acid hybridization

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blotting and nucleic acid hybridization is described. The method utilizes antibodies conjugated with alkaline phosphatase or nucleotide probes complexed with alkaline phosphatase. Then the alkaline phosphatase takes part in a reaction by releasing D -luciferin (Photinus pyralis) from D -luciferin-O-phosphate. Liberated D -luciferin reacts with luciferase, ATP and oxygen under light emission. Light is measured using the Argus-100 a photon counting camera system or photographic films. Bound alkaline phosphatase conjugated antibodies or hybridized nucleotide probes can be visualized. The limit of detection is at present 5 to 50 fg of protein (IgG), corresponding, for example to 30 to 300 × 10^?21 mol. This means a much higher sensitivity of the detection system in comparison to systems used at present. Experiments concerning nucleic acid hybridization and visualization of the emitted light by a photon counting camera (Argus-100) are under investigation.     

Fluorescent Properties of Firefly Luciferases and Their Complexes with Luciferin

Fluorescence of luciferases from Luciola mingrelica (single tryptophanresidue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417,Trp-426) was studied. Analysis of quenching of tryptophan fluorescenceshowed that the tryptophan residue conserved in all luciferases is notaccessible for charged quenchers, which is explained by the presence ofpositively and negatively charged amino acid residues in the close vicinityto it. An effective energy transfer from tryptophan to luciferin wasobserved during quenching of tryptophan fluorescence of both luciferaseswith luciferin. From the data on the energy transfer, the distance betweenthe luciferin molecule and Trp-417 (419) in the luciferin–luciferasecomplex was calculated: 11–15 for P. pyralis and 12–17 for L. mingrelica luciferases. The role of the conserved Trp residuein the catalysis is discussed.     

Stereoselective Incorporation of Isoleucine into Cypridina Luciferin in Cypridina hilgendorfii (Vargula hilgendorfii)

The emission of light in the marine ostracod Cypridina hilgendorfii (presently Vargula hilgendorfii) is produced by the Cypridina luciferin-luciferase reaction in the presence of molecular oxygen. Cypridina luciferin has an asymmetric carbon derived from isoleucine, and the absolute configuration is identical to the C-3 position in L-isoleucine or D-alloisoleucine. To determine the stereoselective incorporation of the isoleucine isomers (L-isoleucine, D-isoleucine, L-alloisoleucine, and D-alloisoleucine), we synthesized four ²H-labeled isoleucine isomers and examined their incorporation into Cypridina luciferin by feeding experiments. Judging by these results, L-isoleucine is predominantly incorporated into Cypridina luciferin. This suggests that the isoleucine unit of Cypridina luciferin is derived from L-isoleucine, but not from D-alloisoleucine.     

A novel bioluminogenic assay for α-chymotrypsin

The use of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin as a novel substrate for α-chymotrypsin has been demonstrated. The kinetic parameters determined are K_M = 0.38mmol/L, k_cat = 6.5 s^?1 and k_cat/k_M = 17,100 (L/mols). The test principle of the coupled assay is the release of aminoluciferin by enzymatic cleavage of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin. Aminoluciferin is oxidized, with light emission, by firefly luciferase (Photinus pyralis) and can be quantified in a luminometric assay. The detection limit for chymotrypsin was found to be 0.3 ng per assay. 6-(N-acetyl-L -phenylalanyl)-aminoluciferin has been synthesized as an example for a new class of highly sensitive substrates. By modification of the peptide residue these new substrates may be suitable for ultrasensitive detection of different proteinases.     

Evolution of beetle bioluminescence: the origin of beetle luciferin

Bioluminescence, the conversion of chemical energy into light in living organisms, is dependent on two principal components, an enzyme luciferase and the substrate luciferin. In beetles, the enzyme luciferase has been extensively studied, with signi?cant enzymological, sequence and structural data now available. Furthermore, the enzyme has been employed in a remarkable number of important applications, from microbial detection and medical imaging to GM gene expression studies. However, there is little information regarding the biosynthesis of beetle luciferin, and here we review the literature and speculate as to its evolutionary origins. Luciferin consists of a benzothiazole moiety attached to a thiazole carboxylic acid moiety, the former being rarely observed in nature but the latter being observed in a broad range of biologically derived molecules. Benzothiazoles are, however, observed in melanogenesis and we speculate as to whether this may be relevant to the understanding of luciferin biosynthesis in beetles. This review examines recent novel insights into beetle luciferin recycling and we assess a range of possible biosynthetic mechanisms. Copyright © 2004 John Wiley & Sons, Ltd.     

Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays

The periodate-mediated conjugation of horseradish peroxidase to antibody is one of the most popular methods to prepare conjugates for enzyme immunoassays of antigens or corresponding antibodies. A very simple method to obtain peroxidase, which is both about five times cheaper than the rather expensive commercial preparations and has a significant higher activity, is reported. Moreover, the conjugation method was critically investigated and considerably simplified. Conjugates thus obtained are about three times more active than the best obtained with the original method.     

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.     

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.     

Interaction of dimethyl-and monomethyloxyluciferin with recombinant wild-type and mutant firefly luciferases

Dissociation constants (Ks) in the pH range 6.5-9.0 for complexes of luciferin, dimethyloxyluciferin (DMOL), and monomethylluciferin (MMOL) with recombinant wild-type and mutant (His433Tyr) luciferases from the Luciola mingrelica firefly were determined by fluorescent titration. The protonated effectors were bound by the wild-type and mutant luciferases better than the nonprotonated ones. The affinity of DMOL for the mutant luciferase was higher than for the wild-type luciferase at alkaline pH, whereas the affinity of MMOL was higher at all pH values studied. The fluorescence emission and excitation spectra of DMOL and MMOL in buffer solution (pH 7.8) were obtained in the absence and presence of luciferase. The fluorescence maxima of DMOL and MMOL complexes with luciferase were 20 and 100 nm, respectively, shifted to shorter wavelengths as compared to the values in buffer solution. This was explained by nonspecific and specific influence of the protein microenvironment on the fluorescence spectra of DMOL and its specific influence on the MMOL fluorescence spectra.     

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.     

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).     

Quenching of the fluorescence of Tyr and Trp residues of firefly luciferase from <Emphasis Type="Italic">Luciola mingrelica</Emphasis> by the substrates

Luciferase of the firefly Luciola mingrelica is characterized by fluorescence of not only the unique Trp residue (lambda(em) = 340 nm), but also that of Tyr residues (lambda(em) = 308 nm). Quenching of the intrinsic fluorescence of the luciferase by its substrates luciferin and ATP (AMP) has been studied. Luciferin (LH2) quenches Trp fluorescence more efficiently than the fluorescence of Tyr residues. Two centers of quenching of Tyr fluorescence by ATP have been found corresponding apparently to the allosteric and active sites of the luciferase with K(s(ATP)) = 20 and 110 microM, respectively. The influence of one substrate on the affinity of luciferase to the second was investigated using fluorescence. ATP (AMP) binding to the allosteric sites of the luciferase significantly affects the affinity of luciferase to LH2. Formation of the complex between the luciferase and LH2 affects the affinity of both allosteric and active sites of the luciferase to ATP (AMP). The observed effects are probably connected with conformational changes in the luciferase molecule upon its interaction with the substrates.     

COMPARATIVE STUDY OF LUMINESCENT AND NONLUMINESCENT STRAINS OF GONYAULAX EXCAVATA (PYRRHOPHYTA)1,2

Three of ten cultures of Gonyaulax excavata (Braarud) Balech isolated from the 1972 New England red tide are nonluminescent, Biochemical components of dinoflagellate bioluminescence were not detected in the extracts from these three isolates. Cells of the nonluminescent cultures were identical to those of luminescent cultures as compared by light microscopy, major body plate tabulation, cell size and growth. Both luminescent and nonluminescent cells were toxic as determined by using the mouse bioassay for paralytic shellfish poisoning. All the 122 clones made from one of the luminescent isolates were luminescent suggesting this feature is a stable trait. We conclude that these isolates represent luminescent and luminescent strains of G. excavata. This is the first intraspecific investigation of in vitro bioluminescent components between nonluminescent and luminescent strains of a dinoflagellate.     

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.     

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.     

The widespread occurrence and tissue distribution of the imidazolopyrazine luciferins

Bioluminescence has been reported to occur in 17 phyla and at least 700 genera. However, the luciferin chemistry of the majority of luminous organisms has yet to be determined. The most common chemistry which is known to occur in deep sea bioluminescence is imidazolopyrazine bioluminescence. The main aim of this study was to examine the phyletic and tissue distribution of imidazolopyrazine luciferins. This will facilitate analysis of imidazolopyrazine bioluminescence at the cellular and molecular levels and, in particular, how and when its chemistry is controlled and expressed in vivo. Assays for both known imidazolopyrazines were established and a range of fresh organisms and tissue were analysed, i.e. fish, cephalopods, copepods, ostracods, amphipods and euphausiids. The main findings were that the number of genera in which coelenterazine has been detected has been increased from 52 to about 90. Also, for the first time, the other known imidazolopyrazine luciferin,Vargula-type luciferin, was quantified in the ostracod Cypridina dentata, but was not detected in any of its potential predators. Neither imidazolopyrazine luciferin was found in several luminous stomiiform fish assayed. Coelenterazine was measured in the livers and photophores of a number of cephalopods and it is apparent that coelenterazine is responsible for both modes of luminescence. © 1997 John Wiley & Sons, Ltd.     

Superoxide anion reacts with enzyme intermediate in the bacterial luciferase reaction competitive with intramolecular electron transfer

Addition of KO₂ in dimethyl sulfoxide (DMSO) to the in vitro bacterial luciferase reaction subsequent to its initiation resulted in a biphasic decay of light emission. The first and more rapid phase is attributed to quenching by DMSO. With DMSO alone the continuing decay is kinetically the same as in a control reaction. With KO₂ added the second decay phase is more rapid and dependent on the KO₂ concentration. The enhanced decay is attributed to superoxide anion generated from KO₂ reacting without light emission with an enzyme peroxy intermediate, breaking down of the peroxide bond through intermolecular electron transfer from the superoxide anion, in competiton with an intramolecular electron transfer from the N(5) position of the flavin ring, which normally leads to the production of the excited luciferase-dihydroflavin-4a-hydroxide. © 1997 John Wiley & Sons, Ltd.     

Emerging tools for bioluminescence imaging

Bioluminescence (BL) relies on the enzymatic reaction between luciferase, a substrate conventionally named luciferin, and various cofactors. BL imaging has become a widely used technique to interrogate gene expression and cell fate, both in small and large animal models of research. Recent developments include the generation of improved luciferase–luciferin systems for deeper and more sensitive imaging as well as new caged luciferins to report on enzymatic activity and other intracellular functions. Here, we critically evaluate the emerging tools for BL imaging aiming to provide the reader with an updated compendium of the latest developments (2018–2020) and their notable applications.     

Establishment and clinical application of enzyme immunoassays for determination of luteinizing hormone releasing hormone and metastin.

Metastin, a 54-residue peptide, was identified as the cognate ligand of human G-protein-coupled receptor GPR54. Since metastin is a gene product of the human metastasis suppressor gene 'KiSS-1', early studies on metastin were focused on its activity as a tumor metastasis suppressor. Recently, there have been some reports that metastin is found in human plasma and is particularly abundant in the plasma of pregnant women. Dysfunction of the GPR54 receptor causes diseases that are characterized by an insufficient release of gonadotropin and lack or delay of pubertal maturation. This information strongly suggests that metastin is involved in the regulation of reproductive endocrine functions. In order to determine the plasma levels of metastin and luteinizing hormone releasing hormone (LHRH) in an isolated hypogonadotropic hypogonadism (IHH) patient, who received intermittent administrations of LHRH, we tried to establish a sensitive and specific enzyme immunoassay. The plasma LHRH levels of the patient were very high, while plasma metastin levels were at almost the same levels as circadian rhythms of healthy male humans. In the central nervous system, metastin stimulates the neuroendocrine reproductive axis. However, the effects of peripheral metastin are not known. Our result suggested that peripheral metastin had a genesis and activity different from central metastin.