Luciferase
Encyclopedia
Luciferase is a generic term for the class of oxidative enzyme
s used in bioluminescence
and is distinct from a photoprotein
. One famous example is the firefly luciferase from the firefly
Photinus pyralis
. "Firefly luciferase" as a laboratory reagent usually refers to P. pyralis luciferase although recombinant
luciferases from several other species of fireflies are also commercially available. The name is derived from Lucifer
, the root of which means 'light-bearer' (lucem ferre).
(a pigment):
The most common luminescent reactions release CO2
as a product. The rates of this reaction between luciferin and oxygen
are extremely slow until they are catalyzed by luciferase, sometimes mediated by the presence of cofactors such as calcium
ions or ATP
. The reaction catalyzed by firefly luciferase takes place in two steps:
Nearly all of the energy input into the reaction is transformed into light. The reaction is 80% to 90% efficient. As a comparison, the incandescent light bulb only converts about 10% of its energy
into light. and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.
, although the enzyme exists in organisms as different as the Jack-O-Lantern mushroom (Omphalotus olearius) and many marine creatures. In fireflies, the oxygen required is supplied through a tube in the abdomen called the abdominal trachea
. The luciferases of fireflies - of which there are over 2000 species
- and of the Elateroidea
(fireflies, click beetles and relatives) in general - are diverse enough to be useful in molecular phylogeny
. The most thoroughly studied luciferase is that of the Photinini
firefly Photinus pyralis, which has an optimum pH of 7.8.
Also well studied is the luciferase from Renilla reniformis
. In this organism, the luciferase is closely associated with a luciferin-binding protein as well as a green fluorescent protein (GFP
). Calcium triggers release of the luciferin (coelenterazine
) from the luciferin binding protein. The substrate is then available for oxidation by the luciferase, where it is degraded to coelenteramide with a resultant release of energy. In the absence of GFP, this energy would be released as a photon of blue light (peak emission wavelength 482 nm). However, due to the closely associated GFP, the energy released by the luciferase is instead coupled through resonance energy transfer to the fluorophore of the GFP, and is subsequently released as a photon of green light (peak emission wavelength 510 nm). The catalyzed reaction is:
Newer luciferases have recently been identified that, unlike Renilla or Firefly luciferase, are naturally secreted molecules. One such example is the Metridia luciferase (MetLuc)that is derived from the marine copepod Metridia longa. The Metridia longa secreted luciferase gene encodes a 24 kDa protein containing an N-terminal secretory signal peptide of 17 amino acid residues. The sensitivity and high signal intensity of this luciferase molecule proves advantageous in many reporter studies. Some of the benefits of using a secreted reporter molecule like MetLuc is its no-lysis protocol that allows one to be able to conduct live cell assays and multiple assays on the same cell.
for a number of purposes. Luciferase gene
s can be synthesized and inserted into organisms or transfected into cells. Mice
, silkworms, and potato
es are just a few organisms that have already been engineered to produce the protein.
In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin
substrate
. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes. This allows observation of biological processes.
In biological research, luciferase commonly is used as a reporter to assess the transcription
al activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest. Luciferase can also be used to detect the level of cellular ATP
in cell viability assays or for kinase activity assays. Additionally proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect caspase
activity and cytochrome P450 activity, among others.
Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice. Different types of cells (e.g. bone marrow stem cells, T-cells) can be engineered to express a luciferase allowing their non-invasive visualization inside a live animal using a sensitive charge-couple device camera (CCD camera).This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models. However, environmental factors and therapeutic interferences may cause some discrepancies between tumor burden and bioluminescence intensity in relation to changes in proliferative activity. The intensity of the signal measured by in vivo imaging may depend on various factors, such as D-luciferin absorption through the peritoneum, blood flow, cell membrane permeability, availability of co-factors, intracellular pH
and transparency of overlying tissue, in addition to the amount of luciferase.
Luciferase can be used in blood bank
s to determine if red blood cell
s are starting to break down. Forensic investigators can use a dilute solution containing the enzyme to uncover traces of blood remaining on surfaces at a crime scene. Luciferase is a heat sensitive protein that is used in studies on protein denaturation, testing the protective capacities of heat shock proteins. The opportunities for using luciferase continue to expand.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s used in bioluminescence
Bioluminescence
Bioluminescence is the production and emission of light by a living organism. Its name is a hybrid word, originating from the Greek bios for "living" and the Latin lumen "light". Bioluminescence is a naturally occurring form of chemiluminescence where energy is released by a chemical reaction in...
and is distinct from a photoprotein
Photoprotein
Photoproteins refer to bioluminescent proteins from luminous organisms. These proteins do not exhibit a luciferin-luciferase reaction, that is, a normal enzyme-substrate reaction. Instead, these proteins display luminescence proportional to the amount of the protein...
. One famous example is the firefly luciferase from the firefly
Firefly
Lampyridae is a family of insects in the beetle order Coleoptera. They are winged beetles, and commonly called fireflies or lightning bugs for their conspicuous crepuscular use of bioluminescence to attract mates or prey. Fireflies produce a "cold light", with no infrared or ultraviolet frequencies...
Photinus pyralis
Photinus pyralis
Photinus pyralis, known by the common names common eastern firefly and big dipper firefly, is the most common species of firefly in North America...
. "Firefly luciferase" as a laboratory reagent usually refers to P. pyralis luciferase although recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
luciferases from several other species of fireflies are also commercially available. The name is derived from Lucifer
Lucifer
Traditionally, Lucifer is a name that in English generally refers to the devil or Satan before being cast from Heaven, although this is not the original meaning of the term. In Latin, from which the English word is derived, Lucifer means "light-bearer"...
, the root of which means 'light-bearer' (lucem ferre).
Chemical reaction
In luminescent reactions, light is produced by the oxidation of a luciferinLuciferin
Luciferins are a class of light-emitting biological pigments found in organisms that cause bioluminescence...
(a pigment):
- luciferin + O2OxygenOxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
→ oxyluciferin + light
The most common luminescent reactions release CO2
Carbon dioxide
Carbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
as a product. The rates of this reaction between luciferin and oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
are extremely slow until they are catalyzed by luciferase, sometimes mediated by the presence of cofactors such as calcium
Calcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
ions or ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
. The reaction catalyzed by firefly luciferase takes place in two steps:
- luciferin + ATPAdenosine triphosphateAdenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
→ luciferyl adenylate + PPiPyrophosphateIn chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...
- luciferyl adenylate + O2OxygenOxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
→ oxyluciferin + AMPAdenosine monophosphateAdenosine monophosphate , also known as 5'-adenylic acid, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid and the nucleoside adenosine. AMP consists of a phosphate group, the sugar ribose, and the nucleobase adenine...
+ light
Nearly all of the energy input into the reaction is transformed into light. The reaction is 80% to 90% efficient. As a comparison, the incandescent light bulb only converts about 10% of its energy
Energy
In physics, energy is an indirectly observed quantity. It is often understood as the ability a physical system has to do work on other physical systems...
into light. and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.
Spectral difference in luciferase bioluminescence
A subtle structural difference in luciferase has been discovered to be the cause of the change in bioluminescence emission colour from a yellow-green to red. The structure of wild-type luciferase and red mutant (S286N) luciferase from the Japanese Genji-Botaru (Luciola cruciata) in complex with an intermediate analogue 5’-O-[N-(dehydroluciferyl)-sulfamoyl] adenosine (DLSA) was examined and studies showed that the wild-type luciferase complexed with DLSA exhibited a ‘closed form’ of the active site, where the side chain of amino acid isoleucine 288 moved towards the benzothiazole ring of DLSA, creating a rigid hydrophobic pocket. The ‘closed form’ wild-type luciferase bound the excited state of oxyluciferin in a highly rigid and nonpolar microenvironment, minimizing energy loss before emitting yellow-green light. The S286N luciferase complexed with DLSA exhibited an ‘open form’ of the active site, where the amino acid side chain of isoleucine 288 did not move towards the benzothiazole ring of DLSA, creating a less rigid and less hydrophobic microenvironment. The ‘open form’ S286N luciferase had a less rigid microenvironment allowing some energy loss from the excited state of oxyluciferin, which resulted in the emission of low-energy red light.Examples
A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The most famous are the firefliesFirefly
Lampyridae is a family of insects in the beetle order Coleoptera. They are winged beetles, and commonly called fireflies or lightning bugs for their conspicuous crepuscular use of bioluminescence to attract mates or prey. Fireflies produce a "cold light", with no infrared or ultraviolet frequencies...
, although the enzyme exists in organisms as different as the Jack-O-Lantern mushroom (Omphalotus olearius) and many marine creatures. In fireflies, the oxygen required is supplied through a tube in the abdomen called the abdominal trachea
Invertebrate trachea
The invertebrate trachea refers to the open respiratory system composed of spiracles, tracheae, and tracheoles that terrestrial arthropods have to transport metabolic gases to and from tissues....
. The luciferases of fireflies - of which there are over 2000 species
Species
In biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...
- and of the Elateroidea
Elateroidea
Elateroidea is a large superfamily of beetles. It contains the familiar click beetles, fireflies, and soldier beetles, and their relatives.Certain clusters of families within the superfamily are more strongly related to one another; for example, the Elateridae has close ties to Anischiidae,...
(fireflies, click beetles and relatives) in general - are diverse enough to be useful in molecular phylogeny
Molecular phylogeny
Molecular phylogenetics is the analysis of hereditary molecular differences, mainly in DNA sequences, to gain information on an organism's evolutionary relationships. The result of a molecular phylogenetic analysis is expressed in a phylogenetic tree...
. The most thoroughly studied luciferase is that of the Photinini
Photinini
The Photinini are a large tribe of fireflies in the subfamily Lampyrinae. Photinus pyralis is famous in biotechnology for its luciferase gene. This is sometimes employed as a marker gene; genetically modified organisms which contain it start to glow like the firefly when brought in contact with...
firefly Photinus pyralis, which has an optimum pH of 7.8.
Also well studied is the luciferase from Renilla reniformis
Sea pansy
The Sea pansy is quite frequently found washed ashore on northeast Florida beaches following northeasterly winds or rough surf conditions. It also can often be found living intertidally completely buried in the sand. Its predator is the striped sea slug, Armina tigrina.The Sea pansy is a...
. In this organism, the luciferase is closely associated with a luciferin-binding protein as well as a green fluorescent protein (GFP
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
). Calcium triggers release of the luciferin (coelenterazine
Coelenterazine
Coelenterazine is the luciferin, the light-emitting molecule, found in many aquatic organisms across seven phyla. It is the substrate in many luciferases and photoproteins including Renilla reniformis luciferase , Gaussia luciferase , aequorin, and obelin.-History:Coelenterazine was simultaneously...
) from the luciferin binding protein. The substrate is then available for oxidation by the luciferase, where it is degraded to coelenteramide with a resultant release of energy. In the absence of GFP, this energy would be released as a photon of blue light (peak emission wavelength 482 nm). However, due to the closely associated GFP, the energy released by the luciferase is instead coupled through resonance energy transfer to the fluorophore of the GFP, and is subsequently released as a photon of green light (peak emission wavelength 510 nm). The catalyzed reaction is:
- coelenterazineCoelenterazineCoelenterazine is the luciferin, the light-emitting molecule, found in many aquatic organisms across seven phyla. It is the substrate in many luciferases and photoproteins including Renilla reniformis luciferase , Gaussia luciferase , aequorin, and obelin.-History:Coelenterazine was simultaneously...
+ O2OxygenOxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
→ coelenteramide + CO2Carbon dioxideCarbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
+ photon of light
Newer luciferases have recently been identified that, unlike Renilla or Firefly luciferase, are naturally secreted molecules. One such example is the Metridia luciferase (MetLuc)that is derived from the marine copepod Metridia longa. The Metridia longa secreted luciferase gene encodes a 24 kDa protein containing an N-terminal secretory signal peptide of 17 amino acid residues. The sensitivity and high signal intensity of this luciferase molecule proves advantageous in many reporter studies. Some of the benefits of using a secreted reporter molecule like MetLuc is its no-lysis protocol that allows one to be able to conduct live cell assays and multiple assays on the same cell.
Applications
Luciferase can be produced in the lab through genetic engineeringGenetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
for a number of purposes. Luciferase gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s can be synthesized and inserted into organisms or transfected into cells. Mice
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
, silkworms, and potato
Potato
The potato is a starchy, tuberous crop from the perennial Solanum tuberosum of the Solanaceae family . The word potato may refer to the plant itself as well as the edible tuber. In the region of the Andes, there are some other closely related cultivated potato species...
es are just a few organisms that have already been engineered to produce the protein.
In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin
Luciferin
Luciferins are a class of light-emitting biological pigments found in organisms that cause bioluminescence...
substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes. This allows observation of biological processes.
In biological research, luciferase commonly is used as a reporter to assess the transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
al activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest. Luciferase can also be used to detect the level of cellular ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
in cell viability assays or for kinase activity assays. Additionally proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect caspase
Caspase
Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis , necrosis, and inflammation....
activity and cytochrome P450 activity, among others.
Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice. Different types of cells (e.g. bone marrow stem cells, T-cells) can be engineered to express a luciferase allowing their non-invasive visualization inside a live animal using a sensitive charge-couple device camera (CCD camera).This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models. However, environmental factors and therapeutic interferences may cause some discrepancies between tumor burden and bioluminescence intensity in relation to changes in proliferative activity. The intensity of the signal measured by in vivo imaging may depend on various factors, such as D-luciferin absorption through the peritoneum, blood flow, cell membrane permeability, availability of co-factors, intracellular pH
Intracellular pH
All cells contain an intracellular fluid whose pH value is known as the intracellular pH . The pHi plays a critical role in the function of the cell, and close regulation is required for cells to survive...
and transparency of overlying tissue, in addition to the amount of luciferase.
Luciferase can be used in blood bank
Blood bank
A blood bank is a cache or bank of blood or blood components, gathered as a result of blood donation, stored and preserved for later use in blood transfusion. The term "blood bank" typically refers to a division of a hospital laboratory where the storage of blood product occurs and where proper...
s to determine if red blood cell
Red blood cell
Red blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...
s are starting to break down. Forensic investigators can use a dilute solution containing the enzyme to uncover traces of blood remaining on surfaces at a crime scene. Luciferase is a heat sensitive protein that is used in studies on protein denaturation, testing the protective capacities of heat shock proteins. The opportunities for using luciferase continue to expand.