Luciferase is a general term for enzymes catalyzing visible light emission by living organisms bioluminescence. A luminometer ab2350 with phelios software atto, tokyo, japan was used. Additionally, luciferase reactions are classified as having either flash or glow kinetics, which have specific detection sensitivities and emission duration times to. The journal of biological chemistry 0 1993 by the american society for biochemistry and molecular biology, inc vol,268, no.
In order to show that atp is an indicator of proliferation andor cell cytotoxicity the assay was carried out under addition of. Firefly luciferase is the lightemitting enzyme responsible for the bioluminescence of fireflies and click beetles. Therefore, we strongly recommend that you follow the procedure below to determine the optimal time after luciferin injection in which to image your model. Thermostabilization of firefly luciferase by in vivo directed evolution. Similar to firefly luciferase, the luminescent reaction. A photinus pyralis and luciola italica chimeric firefly. The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect proteinprotein interactions in vitro, in cellulo, and in vivo. Pdf kinetics of inhibition of firefly luciferase by oxyluciferin and. The luciferase isolated from the firefly photinus pyralis ppy catalyzes a twostep reaction that results in the oxidation of dluciferin accompanied by emission of yellowgreen light with a peak at 560 nm. Pdf kinetics of inhibition of firefly luciferase by.
Kinetics2015 is a guibased computer program for windows xpvista78 to derive chemical kinetic rate models from reaction data. We found that fluc activity in solution has a lower michaelis constant k m for luciferin, lower temperature dependence, and. There are two main types of luminometric atp assays. Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyladenylate. Knowledge about points such as these can give deep insight into the detailed molecular mechanisms behind elementary reactions. Kinetics of the firefly luciferase catalyzed reactions biochemistry. All data and program code are available on figshare at. Kinetiscope is a scientific software tool that provides the bench scientist with an easytouse, rapid, interactive method for the accurate simulation of chemical reactions. Michaelismenten model of enzyme kinetics, demonstrating reaction rate is dependent on the concentration of a substrate s.
The gaussia glow assay kit reagent yields a gaussia dura luciferase reaction with glowtype kinetics and is recommended for luminometers without injectors or for applications requiring batch processing of samples. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length. S imulates acidbase titrations utilizing the same underlying simulation technology used for general reactions. We find that ionizing radiation, in the form of a proton beam from a cyclotron, will eliminate dissolved oxygen prior to any damage to other substrates or to the protein. A realtime kinetic study of luciferase inactivation by pulsed. These features make possible direct correlations between the kinetic. Flexible and easy protocol setup and data processing with pc software. Luciferase genes can be synthesized and inserted into organisms or transfected into cells. Kinetics of the firefly luciferase catalyzed reactions. Interaction of bacterial luciferase with aldehyde substrates and inhibitors received for publication, may 20, 1993, and in revised form, july 12, 1993. The enzyme catalyses the oxidation of firefly luciferin, requiring oxygen and atp. Inject luciferin into animal by an intraperitoneal i.
In the dlr assay chemistry, the kinetics of the renilla luciferase reaction provide a stabilized luminescent signal that decays slowly over the course of the measurement figure 2. Although it has been suggested that kinetics of light production in the. Simulates reaction kinetics utilizing a reaction rate model with userdefined parameters based on a timedriven simulation algorithm, and displays the reactantproduct compositions graphically as a function of time. Figure 7 illustrates the flashtype reaction kinetics of gaussia, cypridina, renilla. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. For the measurement of luciferase luxab kinetic reactions, we. The kinetics, also called reaction kinetics or chemical kinetics, investigates the rates of chemical processes and allows for the determination of reaction rates. Kuzmic 1996 analytical biochemistry 237, 260273 is used for analysis and simulation of chemical and biochemical data. Random mutagenesis by errorprone polymerase chain reaction and mutant library construction.
Chemreax a chemical reaction modeling and simulation app. The firefly luciferase hts assay sct150 is designed with a proprietary mixture of substances that modify the enzymatic. The luciferase assay system yields linear results over at least eight orders of magnitude. A realtime kinetic study of luciferase inactivation by pulsed ionizing radiation. Thermostabilization of firefly luciferase by in vivo. The heart of the software is an optimized simulation engine, providing realtime visual feedback as the user scrolls model parameters such as reaction rate.
Gaussia luciferase assays and vectors thermo fisher. It has broad applications across various fields of cell and molecular biology wherever you want to measure or track expression of a cloned gene. Firefly luciferase luc is the most studied of the luciferase enzymes and the mechanism and kinetics of the reactions catalyzed by this enzyme have been relatively well characterized. It also takes the factors that control these rates into consideration. Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells jolene m. Luciferase enzymes with flash kinetics have maximum sensitivity because of high signal intensity, although the emitted light also rapidly decays. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Quantum yield qy was significantly correlated with luminescence spectrum. Kinetic constants table 1 were determined using the nonlinear least squares method of the enzyme kinetics pro software syntex, which fits data from the michaelismenten equation to a.
Use of fiber optic technology to measure the effects of anesthesia. Chemical modeling software for regression and optimization of chemical kinetics simulation of combustion, nuclear, enzyme, catalyst reactor cstr and other processes and reactions. Indepth characterization of firefly luciferase as a. A realtime kinetic study of luciferase inactivation by. Additionally, luciferase reactions are classified as having either flash or glow kinetics, which have specific detection sensitivities and emission duration times to accommodate different experimental designs. Whether youre in student accommodation, event management, catering or the finance department, we guarantee to take you, your team and your organisation. Stoppedflow kinetic analysis of the bacterial luciferase. Luciferase catalyzes the formation of visible light, fmn, and a carboxylic acid from fmnh2, 02, and the corresponding aldehyde. The light production resulting from the luciferase reaction leads to formation of suicidal adenyloxyluciferin at the enzyme surface. Uptake kinetics and biodistribution of c14dluciferina radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction. Comparative analysis of dualluciferase assay technologies in a.
In the case of firefly luciferase, dluciferin is catalyzed to. Kinetic analysis and modeling of firefly luciferase as a. Firefly luciferase has proven to be a highly sensitive and quantitative reporter gene for studying gene delivery and regulation, and its recent use in live cells and organisms promises to further expand its utility. The reactions of firefly luciferase can be described using. Using a luminometric atp assay with firefly luciferase and luciferin is the most common method for doing quantitative atp measurements. Determining the luciferin kinetic curve for your model. Additionally, there is already considerable knowledge about the reaction mechanism for luciferase.
In circadian research, fluc is frequently expressed under the control of circadian regulated promoter sequences to report oscillations in transcriptional activity, for example, per2. The bioluminescence produced by luciferase, a firefly enzyme, requires three substrates. Kinetic analysis of chemical reactions, simulation. Nonsteady state analysis of enzyme kinetics in real time elucidates substrate association and dissociation rates. Conversely, enzymes with glow kinetics are less sensitive but stably emit light for at least 60 minutes. It results in very short halflife of the light emission with a flashtype kinetics. Renee dale, yuki ohmuromatsuyama, hiroshi ueda, naohiro kato. Reconstructing promoter activity from lux bioluminescent reporters.
The superlight tm luciferase reporter gene assay is based on the quantitation of luciferase expression in mammalian, yeast or e. Comparison of flash and glow atp assays with varioskan. Fluc is the bestcharacterized bioluminescent protein and is used widely as a tool for reporting gene expression levels within living cells gelmini et al. Light production in the presence and absence of these inhibitors 0. Although bio distribution of injected luciferin is rapid, the kinetics of signal production can vary as they may be tissue dependent. Maximum signal intensity and duration is dependent on individual luciferase reaction kinetics. Anesthesia had a marked effect on luciferase reaction kinetics. Firefly luciferase catalyzes the reaction of dluciferin with o2 to produce light. Deuterium kinetic isotope effects and the mechanism of the bacterial luciferase reaction wilson a. Features include a preprocessing section to adjust baselines, smooth the data, and cull excess points. Uptake kinetics and biodistribution of 14 cdluciferina radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction.
Both kinetic and thermodynamic data multiple simultaneous equilibria and be simulated andor fitted by using nonlinear leastsquares regression. Uptake kinetics and biodistribution of 14cdluciferina. The inhibition mechanisms of the firefly luciferase luc by three of the most important inhibitors of the reactions catalysed by luc, dehydroluciferylcoenzyme a lcoa, dehydroluciferin l and lluciferin llh2 were investigated. The data show that with hacat cell line tested there were significant correlation between increased cell number and atp measured by the luciferin luciferase reaction. Generating a kinetic curve for luciferase activity in your model 1. Because bl intensity depends on photons generated through the dluc luciferase reaction, an approach to increase intracellular levels of dluc could improve the detection sensitivity. Demonstration with analysis of firefly luciferase mutants. Spectra were smoothed using quadratic golaysavitzky filter in fl winlab software. Quantum yields and kinetics of the firefly bioluminescence reaction of beetle luciferases. Due to the unique sensitivity of the luciferase reaction, great care must be. The light output from luciferase reactions can be detected with luminometers that are.
Quantum yields and kinetics of the firefly bioluminescence. The reaction results in light production which can be conveniently measured on a luminometer. Luc catalyzes the bioluminescent reaction involving firefly luciferin dlh 2, adenosine triphosphate atp, magnesium ion and molecular oxygen with the. The luciferase assay system incorporates coenzyme a coa for improved kinetics, allowing greater enzymatic turnover and resulting in increased light intensity that is nearly constant for at least 1 minute. Wait three minutes, and sedate the animal by your method of. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin.
Bioluminescence bl imaging based on dluciferin dluc luciferase reaction allows noninvasive and realtime monitoring of luciferase expressing cells. Kinetic constants were determined using a nonlinear least squares method of the enzyme kinetics pro software syntex, which fits data from the michaelismenten equation to. Luc catalysed reactions should contribute to the strong enzyme. Bioluminescent firefly luciferase assays sigmaaldrich. Kintek explorer is simulation and datafitting software for research and education designed specifically for the field of chemical kinetics. A firefly luciferase dual color bioluminescence reporter. In the luciferase reaction, light is emitted when luciferase acts.
The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stoppedflow spectrophotometry at ph 7 and 25 oc. Nonsteady state analysis of enzyme kinetics in real time. Pdf kinetics of inhibition of firefly luciferase by dehydroluciferyl. Firefly luciferase catalyzes the reaction of dluciferin with o 2 to produce light in the presence of mg. Uptake kinetics and biodistribution of c14dluciferina. An investigation of the reaction kinetics of luciferase. The image was generated using the program rasmol version 2. The package is available in versions for microsoft windows, apples mac os x and linux, and includes extensive documentation for. Whichever role you play within your organisation, we understand your challenges. It is also used as an indicator for coupled reactions which produce atp as an end product. Because of the requirement of atp, firefly luciferases have been used extensively in biotechnology. Truncated variants of gaussia luciferase with tyrosine. Before you begin the procedure it is a good idea to set up the software and prepare it to capture your kinetic sequence.
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