Does anyone want to know what the horrible substance Luciferase is? Yes, it is real. The -ase suffix indicates an enzyme.
Luciferase is a generic term for the class of oxidative
enzymes that produce
bioluminescence, and is usually distinguished from a
photoprotein. The name was first used by
Raphaël Dubois who invented the words
luciferin and luciferase, for the substrate and
enzyme, respectively.
[1] Both words are derived from the Latin word
lucifer, meaning "lightbearer", which in turn is derived from the Latin words for "light" (
lux) and "to bring or carry" (
ferre).
[2]
Firefly luciferase
Structure of
Photinus pyralis firefly luciferase.
Luciferases are widely used in
biotechnology, for
microscopy and as
reporter genes, for many of the same applications as
fluorescent proteins. However, unlike fluorescent proteins, luciferases do not require an external
light source, but do require addition of
luciferin, the consumable substrate.
A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The majority of studied luciferases have been found in animals, including
fireflies, and many marine animals such as
copepods,
jellyfish, and the
sea pansy. However, luciferases have been studied in luminous fungi, like the
Jack-O-Lantern mushroom, as well as examples in other kingdoms including
luminous bacteria, and
dinoflagellates.
Firefly and click beetle
The
luciferases of fireflies – of which there are over 2000
species – and of the other
Elateroidea (click beetles and relatives in general) are diverse enough to be useful in
molecular phylogeny.
[3] In fireflies, the oxygen required is supplied through a tube in the abdomen called the
abdominal trachea. One well-studied luciferase is that of the
Photinini firefly
Photinus pyralis, which has an optimum pH of 7.8.
[4]
Sea pansy
Also well studied is the
sea pansy,
Renilla reniformis. In this organism, the luciferase (
Renilla-luciferin 2-monooxygenase) 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:
[5]
Copepod
Newer luciferases have recently been identified that, unlike other luciferases, are naturally secreted molecules. One such example is the
Metridia coelenterazine-dependent luciferase (MetLuc,
A0A1L6CBM1) 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.
[6]
Bacterial
Bacterial bioluminescence is seen in Photobacterium species,
Vibrio fischeri, Vibrio haweyi, and Vibrio harveyi. Light emission in some
bioluminescent bacteria utilizes 'antenna' such as
lumazine protein to accept the energy from the primary excited state on the luciferase, resulting in an excited
lulnazine chromophore which emits light that is of a shorter wavelength (more blue), while in others use a yellow fluorescent protein (YFP) with FMN as the chromophore and emits light that is red-shifted relative to that from luciferase.
[7]
Dinoflagellate
Dinoflagellate luciferase is a multi-
domain eukaryote protein, consisting of an
N-terminal domain, and three
catalytic domains, each of which preceded by a helical bundle domain. The
structure of the dinoflagellate luciferase
catalytic domain has been solved.
[8] The core part of the domain is a 10 stranded
beta barrel that is
structurally similar to
lipocalins and
FABP.
[8] The N-terminal domain is
conserved between dinoflagellate luciferase and
luciferin binding proteins (LBPs). It has been suggested that this region may mediate an interaction between LBP and luciferase or their association with the
vacuolar membrane.
[9] The helical bundle domain has a three
helix bundle structure that holds four important
histidines that are thought to play a role in the
pH regulation of the
enzyme.
[8] There is a large pocket in the β-barrel of the dinoflagellate luciferase at pH 8 to accommodate the
tetrapyrrole substrate but there is no opening to allow the substrate to enter. Therefore, a significant conformational change must occur to provide access and space for a
ligand in the active site and the source for this change is through the four N-terminal histidine residues.
[8] At pH 8, it can be seen that the unprotonated histidine residues are involved in a network of
hydrogen bonds at the interface of the helices in the bundle that block substrate access to the
active site and disruption of this interaction by
protonation (at pH 6.3) or by replacement of the histidine residues by
alanine causes a large molecular motion of the bundle, separating the helices by 11Å and opening the catalytic site.
[8] Logically, the histidine residues cannot be replaced by alanine in nature but this experimental replacement further confirms that the larger histidine residues block the active site. Additionally, three Gly-Gly sequences, one in the N-terminal helix and two in the helix-loop-helix motif, could serve as hinges about which the chains rotate in order to further open the pathway to the catalytic site and enlarge the active site.
[8]
A dinoflagellate luciferase is capable of emitting light due to its interaction with its substrate (
luciferin) and the luciferin-binding protein (LBP) in the
scintillon organelle found in dinoflagellates.
[8] The luciferase acts in accordance with luciferin and LBP in order to emit light but each component functions at a different pH. Luciferase and its domains are not active at pH 8 but they are extremely active at the optimum pH of 6.3 whereas LBP binds luciferin at pH 8 and releases it at pH 6.3.
[8] Consequently, luciferin is only released to react with an active luciferase when the scintillon is acidified to pH 6.3. Therefore, in order to lower the pH,
voltage-gated channels in the scintillon
membrane are opened to allow the entry of
protons from a
vacuole possessing an
action potential produced from a mechanical stimulation.
[8] Hence, it can be seen that the action potential in the vacuolar membrane leads to acidification and this in turn allows the luciferin to be released to react with luciferase in the scintillon, producing a flash of blue light.
Mechanism of reaction
All luciferases are classified as
oxidoreductases (
EC 1.13.12.-), meaning they act on
single donors with incorporation of molecular oxygen. Because luciferases are from many diverse
protein families that are unrelated, there is no unifying mechanism, as any mechanism depends on the luciferase and luciferin combination. However, all characterised luciferase-luciferin reactions to date have been shown to require molecular
oxygen at some stage.
Bacterial luciferase
The reaction catalyzed by bacterial luciferase is also an oxidative process:
- FMNH2 + O2 + RCHO → FMN + RCOOH + H2O + light
In the reaction, molecular oxygen oxidizes
flavin mononucleotide and a long-chain aliphatic
aldehyde to an aliphatic
carboxylic acid. The reaction forms an excited hydroxyflavin intermediate, which is dehydrated to the product FMN to emit blue-green light.
[10]
Nearly all of the energy input into the reaction is transformed into light. The reaction is 80%
[11] to 90%
[12] efficient. In comparison, the
incandescent light bulb only converts about 10% of its
energy into light
[13] and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.
[12]
Applications
Luciferases can be produced in the lab through
genetic engineering for a number of purposes. Luciferase
genes can be synthesized and inserted into organisms or
transfected into cells. As of 2002,
mice,
silkworms, and
potatoes are just a few of the organisms that have already been engineered to produce the protein.
[14]
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.
[15] Since light excitation is not needed for luciferase bioluminescence, there is minimal
autofluorescence and therefore virtually background-free fluorescence.
[16] Therefore, as little as 0.02 pg can still be accurately measured using a standard
scintillation counter.
[17]
In biological research, luciferase is commonly used as a reporter to assess the
transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a
promoter of interest.
[18] 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.
[15][18]
Luciferase can also be used to detect the level of cellular ATP in cell viability
assays or for kinase activity assays.
[18][19] Luciferase can act as an ATP sensor protein through
biotinylation. Biotinylation will immobilize luciferase on the cell-surface by binding to a
streptavidin-
biotin complex. This allows luciferase to detect the efflux of ATP from the cell and will effectively display the real-time release of ATP through bioluminescence.
[20] Luciferase can additionally be made more sensitive for ATP detection by increasing the luminescence intensity by changing certain
amino acid residues in the sequence of the protein.
[21]
Whole animal imaging (referred to as
in vivo when living or, otherwise called
ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice.
[22] 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.
[23][24] 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.
[25]
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.
[26]
In November 2021, a White House correspondent for the conservative outlet
Newsmax falsely tweeted that a
COVID-19 vaccine contained luciferase "so that you can be tracked."
[27][28]
https://en.wikipedia.org/wiki/Luciferase
If the story is too good to be true, then a wise journalist can do some fact-checking. Luciferase has nothing to do with any malign spirits. Tracking us? We'd need far more than what it takes to light up a firefly. Seemingly everyone has Internet access, and that is good enough for determining whether an easy and obvious connection between a word and a story does not exist.
Of course, if the government really does want to track you down as is the case for a fugitive, then credit cards, debit cards, food-aid cards, and cell phones can do the trick. You need to be paid under the table, which creates potential trouble for a potential employer who faces charges for tax fraud of various sorts.