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