Highly sensitive simultaneous bioluminescent measurement of acetate kinase and pyruvate phosphate dikinase activities using a firefly luciferase-luciferin reaction and its application to a tandem bioluminescent enzyme immunoassay.

We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03 x 10(-20) and 2.05 x 10(-20) mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample.     

Quantitative and bioluminescent assay to measure efficacy of conventional and DNA vaccinations against Helicobacter pylori

Vaccination against Helicobacter pylori using DNA sequences encoding Urease A and B subunits was compared to immunization with urease antigen and MTP-PE in a liposome formulation. To determine the effectiveness of a vaccine against H. pylori in a mouse model it is essential to quantify the number of H. pylori remaining in the stomachs following challenge with an inoculum of live bacteria. Culture assays and enzymatic assays produce inconsistent results often unsuitable to conclude if vaccine candidates are protective. To overcome this problem, we developed two assays: 1) a competitive quantitative PCR using a colorimetric readout and 2) a non-competitive direct quantitative PCR using a highly sensitive bioluminescent readout. The competitive PCR requires coamplification of a segment of the urease C sequence and an internal control standard in a competitive manner using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, bioluminescent assay measures the amplified DNA directly using a flash-type luminescent tag and a specific probe. The Sydney strain of H. pylori was used for the mouse infection model. Quantification of H. pylori by either the bioluminescent assay or the competitive PCR was reliable, specific and sensitive compared to quantitative growth assays which often gave false results. The bioluminescent assay was much more sensitive and less labor/time intensive than the competitive PCR. The bioluminescent assay was able to quantitate as few as 100 bacteria, while the competitive assay could not detect less than 10(3) bacteria per mouse stomach. Quantification of H. pylori by bioluminescent assay was superior to the competitive assay and may be used for research applications, such as the development of vaccines, pathogenesis of gastric disease and monitoring of antibiotic treatment.     

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop-mediated isothermal amplification coupled to a new bioluminescent assay

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90–120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.     

Bioluminescence flow sensor for determination of creatine kinase activity in blood

A bioluminescent flow sensor was developed for the assay of creatine kinase (CK) using firefly luciferase immobilized on a nylon coil. The CK-catalysed reaction of creatine phosphate with ADP took place in a cuvette before the injection into the bioluminescent detector coil. The response was linear from 0.1 to 100 U l^? at 25°C. An advantage of the flow sensor is a detection limit of less than 0.1 U l^?1, which, together with a high precision, allows determination of the CK activity in blood sera in about 5 min. The intra- and inter-assay reproducibilities (RSD) were less than 10% and the recovery range was 86–110%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.     

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

Background

Luciferases, 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.

Methods

Here, 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.

Results

In 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.

Conclusion

With 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.

     

THE FLUORESCENT PRODUCT OF SCALEWORM BIOLUMINESCENT REACTION: AN IN VITRO STUDY

Abstract— The fluorescence of scaleworms has been attributed by Harvey (1952) to a product of the bioluminescent reaction confined in the scales of these worms. We have purified this fluorescent molecule by gel filtration. This compound has an apparent low molecular weight as shown by polyacrylamide gel clectrophoresis in the presence of SDS. The yield of the fluorescent product, after gel filtration, varies with the stimulation of the bioluminescence, triggered either chemically or enzymatically. The fluorescence spectrum of the purified product is similar to the one observed in vivo , with a maximum centered at 520 nm. Consequently, the fluorescent compound isolated is likely the reaction product of the bioluminescent reaction.     

Does phenomenological kinetics provide an adequate description of heterogeneous catalytic reactions?

Phenomenological kinetics (PK) is widely used in the study of the reaction rates in heterogeneous catalysis, and it is an important aid in reactor design. PK makes simplifying assumptions: It neglects the role of fluctuations, assumes that there is no correlation between the locations of the reactants on the surface, and considers the reacting mixture to be an ideal solution. In this article we test to what extent these assumptions damage the theory. In practice the PK rate equations are used by adjusting the rate constants to fit the results of the experiments. However, there are numerous examples where a mechanism fitted the data and was shown later to be erroneous or where two mutually exclusive mechanisms fitted well the same set of data. Because of this, we compare the PK equations to "computer experiments" that use kinetic Monte Carlo (kMC) simulations. Unlike in real experiments, in kMC the structure of the surface, the reaction mechanism, and the rate constants are known. Therefore, any discrepancy between PK and kMC must be attributed to an intrinsic failure of PK. We find that the results obtained by solving the PK equations and those obtained from kMC, while using the same rate constants and the same reactions, do not agree. Moreover, when we vary the rate constants in the PK model to fit the turnover frequencies produced by kMC, we find that the fit is not adequate and that the rate constants that give the best fit are very different from the rate constants used in kMC. The discrepancy between PK and kMC for the model of CO oxidation used here is surprising since the kMC model contains no lateral interactions that would make the coverage of the reactants spatially inhomogeneous. Nevertheless, such inhomogeneities are created by the interplay between the rate of adsorption, of desorption, and of vacancy creation by the chemical reactions.     

Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device

A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.     

A facile electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases

Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective electrophoretic technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.     

Bioluminescence and exogenous compounds: physico-chemical basis for bioluminescent assay

Bioluminescent systems are convenient objects to study mechanisms of influence of exogenous molecules on living organisms. Classification of physical and physico-chemical mechanisms of the effects of luminous bacteria Photobacterium leiognathi on bioluminescent reactions is suggested. Five mechanisms are discussed: (1) change of electron-excited states' population and energy transfer, (2) change of efficiency of S-T conversion in the presence of external heavy atom, (3) change of rates of coupled reactions, (4) interactions with enzymes and variation of enzymatic activity, (5) nonspecific effects of electron acceptors. Effects of various groups of chemical compounds are discussed according to the classification suggested. The compounds are: a series of fluorescent dyes, organic oxidizers, organic and inorganic heavy-atom containing compounds, and metallic salts. Applications of fluorescence time-resolved and steady-state techniques, as well as bioluminescence kinetics study, are discussed. The patterns of exogenous compounds' influence form a physico-chemical basis for bioluminescent ecological assay.     

Electrochemical study of the effect of ADP and AMP on the kinetics of glutamate dehydrogenase

A chronoamperometric method based on the 'diffusion' layer concept of the convective system was used to assay the glutamate dehydrogenase (GLDH) activity. Once the reaction was initiated by adding the enzyme GLDH into a well-stirred nicotinamide adenine dinucleotide (NADH, coenzyme) solution, the steady-state oxidation limiting current of NADH would decrease linearly in a short time. The major advantage of this method is that it directly indicates the continuous in-situ change of the coenzyme concentration, thus, the real initial reaction rate of enzyme-catalyzed reaction, V0, can be determined. Using this method, the effect of adenosine-5'-monophosphate (AMP) and adenosine-5'-diphosphate (ADP) on the GLDH activity has been monitored. The results showed that ADP and AMP could increase the activity of GLDH. This activation mechanism was proposed by the voltammetric study.     

A Highly Sensitive Bioluminescent Assay of β-D-Galactosidase from Escherichia coli Using 2-Nitrophenyl-β-D-galactopyranoside as a Substrate

Abstract

A highly sensitive bioluminescent assay of β-D-galactosidase from Escherichia coli is described. D-Galactose was released from 2-nitrophenyl-β-D-galactopyranoside as a substrate by the catalytic action of β-D-galactosidase, and subsequently NADH was formed using galactose dehydrogenase. NADH was measured by a bioluminescent assay using NAD(P)H:FMN oxidoreductase and luciferase from Photobacterium fischeri. The detection limits of β-D-galactosidase for 100 and 1,000 min assays were 2 × 10^?21 mol and 2 × 10^?22 mol, respectively. When the volume of the reaction mixture for β-D-galactosidase assay was reduced from 2 μ to 0.5 μ1, the detection limits were reduced to half.     

Improved detection of toxic chemicals using bioluminescent bacteria

A sensitive, rapid and simple bioluminescent (BL) assay using bioluminescent bacteria to detect the toxic activity of several chemicals is described. This assay is based on the measurement of inhibition of light production of a bioluminescent bacterial strain, isolated from seawater, in the presence of different toxins like heavy metals, organic chemicals, such as benzene, toluene, ethylbenzene, xylene (BTEX) and a wide range of pesticides in environmental samples. The improvement with respect to other commercial and non-commercial bioluminescent assays consists of the possibility to work at room temperature without the need to thermostat, thus allowing the use of simpler and low cost instruments, or to improve the assay using a microplate format, which makes it possible to analyse several samples also continuously for several hours. Using lyophilised bacteria, the assay is performed in less than an hour, without any bacterial cultivation, which makes the test suitable for rapid and sensitive evaluation of chemical pollutants in environmental samples.     

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM02734, a novel antineoplastic agent, in dog plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel antineoplastic agent, PM02734, in dog plasma. The method was validated to demonstrate the specificity, limit of quantification (LOQ), accuracy, and precision of measurements. The calibration range for PM02734 was established using PM02734 standards from 0.05 to 100 ng/mL in blank plasma. The dominating ions were doubly charged molecular ions [M+2H]2+ at m/z 740.0 instead of singly charged ones at m/z 1478.4. The selected reaction monitoring (SRM), based on the m/z 740.0 --> 212.2 transition, was specific for PM02734, and that based on the m/z 743.8 --> 212.2 transition was specific for deuterated PM02734 (the internal standard, IS); no endogenous materials interfered with the analysis of PM02734 and IS from blank plasma. The assay was linear over the concentration range 0.05-100 ng/mL. In terms of sensitivity of assay 0.05 ng/mL is a very low LLOQ, especially considering PM02734 is a peptide. The correlation coefficients for the calibration curves ranged from 0.9990 to 0.9999. The mean intraday and interday accuracies for all calibration standards (n = 9) ranged from 93 to 111% (< or =11% bias) in dog plasma, and the mean interday precision for all calibration standards was less than 6.4%. The mean intra- and interday assay accuracy for all quality control replicates in dog plasma (n = 9), determined at each QC level throughout the validated runs, ranged from 85-111% (< or =15% bias) and from 99-109% (< or =9% bias), respectively. The mean intra- and interday assay precision was less than 12.1 and 13.3% for all QC levels, respectively. The assay has been used to support preclinical pharmacokinetic (PK) and toxicokinetic studies. The results showed that preclinical samples could be monitored for PM02734 up to 168 h after dosing, which allowed us to identify multiple elimination phases and accurately estimate PK information.     

Bioluminescent Enzymatic Assay as a Tool for Studying Antioxidant Activity and Toxicity of Bioactive Compounds

A bioluminescent assay based on a system of coupled enzymatic reactions catalyzed by bacterial luciferase and NADH:FMN‐oxidoreductase was developed to monitor toxicity and antioxidant activity of bioactive compounds. The assay enables studying toxic effects at the level of biomolecules and physicochemical processes, as well as determining the toxicity of general and oxidative types. Toxic and detoxifying effects of bioactive compounds were studied. Fullerenols, perspective pharmaceutical agents, nanosized particles, water‐soluble polyhydroxylated fullerene‐60 derivatives were chosen as bioactive compounds. Two homologous fullerenols with different number and type of substituents, C₆₀O_2–4(OH)_20–24 and Fe_0.5C₆₀(OH) _xO_y (x + y = 40–42), were used. They suppressed bioluminescent intensity at concentrations >0.01 g L^?1 and >0.001 g L^?1 for C₆₀O_2–4(OH)_20‐24 and Fe_0.5C₆₀(OH)_xO_y, respectively; hence, a lower toxicity of C₆₀O_2–4(OH)_20–24 was demonstrated. Antioxidant activity of fullerenols was studied in model solutions of organic and inorganic oxidizers; changes in toxicities of general and oxidative type were determined; detoxification coefficients were calculated. Fullerenol C₆₀O_2–4(OH)_20–24 revealed higher antioxidant ability at concentrations 10^?17?10^?5 g L^?1. The difference in the toxicity and antioxidant activity of fullerenols was explained through their electron donor/acceptor properties and different catalytic activity. Principles of bioluminescent enzyme assay application for evaluating the toxic effect and antioxidant activity of bioactive compounds were summarized and the procedure steps were described.     

Luciferin Regeneration in Firefly Bioluminescence via Proton-Transfer-Facilitated Hydrolysis,Condensation and Chiral Inversion

Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2-cyano-6-hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L-cysteine in vivo to form L-luciferin via a condensation reaction; and L-luciferin inverts into D-luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long-term flashing of fireflies without an in vitro energy supply.     

Current Status of Research on Fungal Bioluminescence: Biochemistry and Prospects for Ecotoxicological Application

Over the last half decade the study of fungal bioluminescence has regained momentum since the involvement of enzymes has been confirmed after over 40 years of controversy. Since then our laboratory has worked mainly on further characterizing the substances involved in fungal bioluminescence and its mechanism, as well as the development of an ecotoxicological bioluminescent assay with fungi. Previously, we proved the involvement of a NAD(P)H‐dependent reductase and a membrane‐bound luciferase in a two‐step reaction triggered by addition of NAD(P)H and molecular oxygen to generate green light. The fungal luminescent system is also likely shared across all lineages of bioluminescent fungi based on cross‐reaction studies. Moreover, fungal bioluminescence is inhibited by the mycelium exposure to toxicants. The change in light emission under optimal and controlled conditions has been used as endpoint in the development of toxicological bioassays. These bioassays are useful to better understand the interactions and effects of hazardous compounds to terrestrial species and to assist the assessment of soil contaminations by biotic or abiotic sources. In this work, we present an overview of the current state of the study of fungal luminescence and the application of bioluminescent fungi as versatile tool in ecotoxicology.     

Effect of heavy atoms in bioluminescent reactions

Bioluminescent reactions of luminous organisms are excellent models for studying the effects of heavy atoms on enzymatic processes. The effects of potassium halides with halide anions of different atomic weight were compared in bioluminescent reactions of the firefly (Luciola mingrelica), a marine coelenterate (Obelia longissima), and a marine bacterium (Photobacterium leiognathi). Two mechanisms of the effects of the halides were examined—the physicochemical effect of the external heavy atom, based on spin–orbit interactions in electron-excited structures, and the biochemical effect, i.e. interactions with the enzymes resulting in changes of enzymatic activity. The physicochemical effect was evaluated by using photoexcitation of model fluorescent compounds (flavin mononucleotide, firefly luciferin, and coelenteramide) of similar structure to the bioluminescence emitters. The bioluminescent and photoluminescent inhibition coefficients were calculated and compared for the luminous organisms to evaluate the relative contributions of the two mechanisms. The biochemical mechanism was found to be dominant. Hence, the bioluminescent reactions can be used as assays to monitor enzyme inhibition, in metabolic processes, by Br or I-containing compounds.     

Allosteric Regulation of Enzymatic Reactions in a Transparent Inorganic Sol-Gel Material

Glutamate dehydrogenase is encapsulated in a transparent porous silicate matrix by using sol-gel techniques. The inorganic polymer is formed around the enzyme (MW > 300,000 D). The enzyme is active in the material, catalyzes the reaction of L-glutamate to 2-oxoglutarate and follows Michaelis-Menten kinetics. The allosteric regulators ADP and GTP inhibit or activate the reaction; at pH 6, GTP acts as a strong activator and ADP acts as an inhibitor. This system involves a complex series of interactions; the co-enzyme NAD⁺ is required for catalysis, large-scale conformational changes accompany the binding of the substrate and coenzyme to the enzyme, the activators/inhibitors must bind to the enzyme to regulate the reactions, and the substrates and products must diffuse through the matrix to and from the binding site. The influence of the unique matrix on the complex enzymatic system is discussed.