Luciferin

Luciferins are different natural substances that are used in a variety of bioluminescent organisms to produce light. By catalytic activity of the corresponding luciferase enzyme to react with oxygen ( oxidation). In changing, usually the separation of sub-groups of the luciferin, energy is released as light. Both luciferins and luciferases are art- or taxonspezifisch, so characteristic of every living creature group.

• 3.1 The firefly luciferin, a benzothiazoles 3.1.1 bioluminescent
• 3.1.2 Reaction Mechanism
• 3.1.3 synthesis
• 3.1.4 Evolutionary origins
• 3.1.5 Luminescence of other insects
• 3.1.6 dehydroluciferin

• 3.2.1 component F in krill

• 3.3.1 lux genes

• 3.4.1 mechanism
• 3.4.2 Watasenia - luciferin
• 3.4.3 Vargula luciferin
• 3.4.4 Dehydrocoelenterazin from Symplectoteuthis oualaniensis

• 4.1 The Latia - luciferin
• 4.2 luciferin from Diplocardia longa

• 5.1 diagnostics
• 5.2 Genetic Engineering / Biotechnology

History

At the beginning of the 18th century, René Reaumur observed that powder of the dried and ground bioluminescent organisms light up when water is added. The first works on the luciferin-luciferase systems date back to the French Raphael Dubois. He discovered when working on fireflies in 1885 that a substance is consumed in a light -giving response. He extracted from different organisms that produced biological light, this material and referred to him as luciferin, as it is not destroyed by heat. The other heat-labile component of the scientist has called luciferase. Today is commonly referred to as a luciferase, the enzyme which converts the associated luciferin. During mixing of luciferin with luciferase in the presence of oxygen Dubois could mimic the natural bioluminescence.

The next studies were conducted by Harvey Newton beginning of the 20th century. He found out that there is a specificity in each luciferin - luciferase system. So luciferins of a species can not be implemented by the luciferase an alien species. Finally, each bioluminescent system requires oxygen. This has been observed already in the 18th century by Robert Boyle.

Properties

Bioluminescent systems are not evolutionarily conserved, the luciferases share no sequence homology. Luciferases occur but in 17 different tribes and at least 700 mostly marine genera. Apparently, they were often "invented" phylogenetic studies indicated that the luciferin-luciferase systems have more than 30 independent origins.

Definition

According to the classical definition, is bound to the luciferase luciferin, the light emitter. The luciferase is under consumption of oxygen luciferin this is, sometimes it also cofactors such as ATP or ions are required. The oxidized luciferin is initially in a state of transition I and then passes - often after decarboxylation and further intermediate steps - in an electronically excited state P *. This falls very short time (a few nanoseconds) back to its ground state and emits P during which a light quantum. Normally, the unreacted luciferins are fluorophores, because they can get into an excited state by irradiation of light.

Principle

To reach the excited state of P *, biochemically much energy is required. The emission of photons with a wavelength of 500 nm (green, energy is about 2 eV / photon) requires about 250 kJ / mol - for comparison, the hydrolysis of ATP to ADP and phosphate is about 30 kJ / mol free. Furthermore, the energy can be released only in one step.

The most common principle is to form a four-membered ring, a dioxetane or dioxetanone ( α - peroxylactone ). After decarboxylation, the electronically excited state forms.

Sometimes, the fluorescence does not match expected, for example in in vitro studies ( in vitro ). There are several causes. How to emit enzymes bound luciferins at different oxidation as free luciferins after excitation by light. Sometimes the energy is transferred to a second fluorophore, as for example happens with aequorin to GFP in Aequorea victoria.

Quantum yield Q

Whether the implementation of a luciferin is efficient by the corresponding luciferase, is determined by the so-called quantum yield Q ( quantum yield ). It is defined as the number of emitted photons per molecule reacted for luciferin. Due definition the maximum value of Q = 1, this would mean that for every molecule of unreacted luciferin, a light quantum is free. The fastest quantum efficiency has been demonstrated for the firefly Photinus pyralis out with Q = 0.41.

Luciferin - types

Luciferin-luciferase systems are found in many species. There are four main classes of the luciferin-luciferase system, wherein the luciferin transported to implementation by a luciferase in an electronically excited state, and thus the actual light emitter.

The firefly luciferin, a benzothiazoles

Bioluminescent insects are represented in the four orders Collembola, Hemiptera, Coleoptera and Diptera. However, only the bioluminescent organisms of the latter two systems were investigated. In Coleoptera ( beetles) representatives of three families can produce light: Phengodidae ( spring Firefly ), Elateridae ( click beetles ) and Lampyridae ( fireflies ).

The American firefly Photinus pyralis ( firefly engl. ) belongs to the family Lampyridae. He has already been used by Dubois to his studies on luciferins luciferases (see above). Scientific studies of the first bioluminescence in P. pyralis were initiated in 1917 by Harvey. Meanwhile, this luciferin-luciferase system, the best studied and is presented below.

Bioluminescent

For the reaction requires a luciferase substrate D- luciferin (LH2 ), a benzothiazole, the consumption of oxygen by. The works of William D. McElroy in the late 1940s have shown that are required for the reaction ATP and magnesium ions as cofactors:

In comparison to other systems, luciferins (see below) is the luciferin from fireflies a relatively stable compound. The melting point is 205-210 ° C. Its molar extinction coefficient at 328 nm, ε = 18,200 M-1 · cm -1. Fluoresces luciferin and shows an emission maximum at lambda max = 537 nm

The luciferase ( EC 1.13.12.7 ) of the Fireflies has a molecular mass of approximately 60-62 kDa in P. pyralis exactly 61 kDa and is composed of 550 amino acids. It catalyzes the oxidative decarboxylation of luciferin to oxyluciferin (oxy -L, see also the figure below, box). The reaction proceeds in the peroxisomes of the light organ cells. The structure of the luciferase from P. pyralis was first presented in 1956 with a resolution of 200 pm. For this analysis, large amounts were collected at fireflies with helpful children who had received one cent reward for each Delivered copy.

Without bound luciferase substrate is in the open conformation; a large N -terminal and a small C-terminal region form a deep furrow. When substrate binding a conformational change leads to closing the furrow. The mid-1980s could luciferase are successfully incorporated into the genome of the bacterium E. coli and expressed. Luciferases from fireflies Lampyridae family are very similar. However, the differences to determine the color of the emitted light. Depending on the emission maximum is lambda max of the released light between 530 nm ( green) and 635 nm ( red).

The reaction proceeds best in vitro from at a pH value of 7.8 and at temperature of 23-25 ​​° C. In vivo, the color of the emitted light is yellow-green to yellow ( 552-582 nm). In the laboratory, the reaction shows a wider range of colors. In acidic medium, the light reddish (615 nm) appears in a neutral medium green-yellow.

Reaction mechanism

The exact reaction mechanism of the reaction is known. By the D- luciferin ATP is first adenylated at the carboxyl group, which is released pyrophosphate as a leaving group (1, see Figure ). Through this activation, the proton on the C4 atom can be abstracted, it forms a carbanion (2). Then the luciferin at the C4 atom can be oxygenated, it forms a linear hydroperoxide (3). This makes the elimination of AMP Dioxetanonring (4). After decarboxylation therefrom oxyluciferin what either as a monoanion ( keto form, 5) or dianion forms ( enol form ) may be present. In both cases, the oxyluciferin in an energetically excited state. It falls under delivery of a photon (red light or yellow- green light) back to its ground state. The oxyluciferin itself has not yet been isolated in pure form, as it is extremely unstable.

The reaction mechanism with formation of a Dioxetanons was unequivocally assigned at the end of the 1970s through the work of Shimomura. This isotopically labeled 18O was used in the reaction ( H218O or 18O2 ). The results of this work replaced the previously postulated hypothesis that oxyluciferin arises by linear bond cleavage. If this really was, the released carbon dioxide would contain an oxygen atom derived from water. In fact, he comes from the oxygen.

Synthesis

As the insects - or microbial symbionts - produce the luciferin, is not fully understood. It is known that D- luciferin is not taken directly from the beetle ( unless in female beetles of the species Photuris that eat their male counterparts ). One possibility is, the oxyluciferin produced after the reaction to recycle light back to luciferin. Here, the first to be converted to oxyluciferin 2-cyano- 6- hydroxybenzothiazole ( 2C6HB ), which is determined by the luciferin - regenerating enzyme ( LRE ), for example, in Photinus pyralis, catalyzed. 2C6HB then condensed with a D- cysteine ​​to D-luciferin. This condensation reaction is also used in the chemical Luciferinsynthese ( see figure right way ).

A recently discussed possibility is, however, assume that 2C6HB first forms L- luciferin with L- cysteine. This is then racemized through intermediate steps to D- luciferin (see also Figure left path ).

Both possibilities of this biosynthesis still have some problems:

So the luciferin - regenerating enzyme is not produced bioluminescent beetles in the light- producing organs. Since oxyluciferin is unstable in aqueous solutions, one would have to just there expect to find LRE in larger quantities. In addition, the reactive 2C6HB not only with cysteine ​​, but also react with other metabolites. It is also not clear where, for example, dates back to the D- cysteine ​​and could be discriminated as between L -cysteine ​​and D -cysteine. From L- cysteine ​​reacts 2C6HB namely also to L- luciferin. Although this can absorb the luciferase substrate, but inhibits the light response. Also could not be verified, the enzyme that catalyzes the racemization of one.

It is also unclear as to the beetles (or symbionts ) Benzothiazolene produce.

Evolutionary origins

Possibly the luciferin - luciferase reaction has developed with fireflies from a completely different biological function out. It is believed that the Luciferinmolekül occurred in a later evolutionary result and led to a light reaction. This is suggested that the luciferase and efficient coenzyme A can condense on the Luciferinmolekül and thus fulfills the function of a classical fatty acid -CoA ligase. The luciferase may be used in this context, fatty acids such as arachidonic acid, which shares similar structural characteristics with the luciferin.

Due to these additional catalytic properties the original luciferase might have been a fatty acid CoA ligase. By the appearance of the luciferin and the associated light reaction, a selective advantage revealed: The Adenylierungsreaktion had prevailed over time. This thesis has been demonstrated on non-luminescent mealworm Tenebrio molitor. This has no luciferin, but fatty acid -CoA ligases. Interestingly, it can be observed by the addition of luciferin and there a light reaction. But without knowledge of how the biosynthesis of Leuchtkäferluciferins expires, a more detailed evolutionary analysis is difficult.

Luminescence of other insects

Even with luminescent insects of the other two families Phenogodidae and Elateroidae the firefly luciferin occurs. Either click beetles (eg, firefly Pyrophorus noctilucus ) is the bioluminescence of spring fireflies ( "railroad worm" eg Phrixothrix, engl. ) With those from fireflies almost identical. But with the spring fireflies do not show only the larvae bioluminescence, the adult animals.

On the other hand have luminescent (Diptera ) on ( Arachnocampa or Orfelia ) nothing in common with the luciferin from fireflies. Is that of the larvae of the North American fungus gnat ( Orfelia fultoni ) emitted light, moreover, the bluest ( lambda max = 460 nm ) which is generated by insects. This, for example, live in the Waitomo Caves.

Dehydroluciferin

In vitro it was shown that enzyme-bound, adenylated D- luciferin (D - LH2 · AMP) can be implemented in a dark reaction. Here it reacts with oxygen to form hydrogen peroxide and dehydroluciferin (L · AMP). The latter can be released from the luciferase by pyrophosphate and causes ATP.

L · AMP is a potent inhibitor of the luciferase. Whether the formation of dehydroluciferin occurs under physiological conditions, is not known. At least the resulting hydrogen peroxide ( H2O2) could be detoxified rapidly in the peroxisomes.

Tetrapyrrole luciferin of dinoflagellates and Euphausiidae

• Bioluminescence of dinoflagellates, caused by the breaking of the waves

Similar uptake ( Manasquan )

Receiving near Carlsbad (California, USA)

Reception at the beach of Seal Beach (California, USA)

The luciferin in this group corresponds to the chemical foundations of a linear tetrapyrrole open and can be found among others in dinoflagellate ( Noctiluca, Gonyaulax, Pyrocystis ). Meeresleuchten, which was previously incorrectly referred to as phosphorescence is largely back to this unicellular algae. The investigations of the luciferin-luciferase system began in the late 1950s to the dinoflagellate Lingulodinium polyedrum ( synonym: Gonyaulax polyedra ) by the work of J. Woodland Hastings and employees.

For light emission in addition to luciferin, and the corresponding a, about 135 kDa luciferase ( LCF), a so-called luciferin binding protein ( luciferin binding protein (LBP) ) is necessary. LBD is a homodimer, one subunit is 72 kDa in size. Luciferin this family is very unstable at low pH values ​​(< pH 4) and high salt already low oxygen concentrations. It has been shown that at pH 8.0 the LBD binds to the dinoflagellate luciferin, but not at pH 6.3. Characterized the substrate is to be protected until the reaction best takes place at pH 6.3, since the luciferase is inactive in a slightly alkaline medium (pH 8.0). For the reaction of luciferin with oxygen has been suggested that this occurs via several intermediates via radicals. The reaction itself takes place in special organelles, called Scintillone. These are, on average, 0.4 uM large and mainly contain luciferases luciferins and LBP. The light that is produced in this reaction, blue -green ( excitation maximum 390 nm, emission maximum of approximately lambda max = 470 nm with lambda max =) appears. The light emitter is an enzyme-bound intermediate of the to -converting luciferin.

It is still unclear today whether the luciferin is derived because of its relationship to chlorophyll a from this or only of several amino acids ( glycine and glutamic acids ) must be built up gradually. Moreover, it is paradoxical that formed during the light reaction oxy - luciferin is not a fluorophore.

Component F in krill

A luciferin with almost identical structure was also observed in lumeszierenden Euphausiidae ( krill ) found, for example, in Meganyctiphanes norvegica Euphausia pacifica or. There, it is indicated as component F, which is obtained from food. The reaction mechanism corresponds to that of dinoflagellates.

Flavin, a bacterial luciferin

Luminescent bacteria utilize reduced flavin mononucleotide ( FMNH2, which is also called riboflavin -5-phosphate ) for a light- forming reaction. They come in either terrestrial ago ( Vibrio and Xenorhabdus ) or free-living in the sea ( Beneckea, Vibrio ). They are also responsible for the bioluminescence of many bright deep-sea fish, where they are kept as symbionts in special light organs ( Photobacterium ). The previously identified bioluminescent bacteria are all Gram- negative. A well-known bacterium with Biolumineszenzeigenschaft example, Vibrio fischeri.

The study of bacterial bioluminescence led in the 1950s to special advances. The researchers Milton J. Cormier and Bernard L. Strehler found four factors are necessary for the bioluminescent reaction: In addition to the FMNH2 a luciferase, molecular oxygen and a long-chain aldehyde of a saturated hydrocarbon is required. The aldehyde, hexadecanal was designated before its chemical identification as "kidney cortex factor" because the aldehyde was isolated from the adrenal cortex of pigs. For the light response, other aldehydes may be used, such as decanal or dodecanal. The following table shows the composition of 40 g of bacteria isolated again. It is believed that mainly tetradecanal is implemented.

FMNH2 and the aldehyde are reacted oxygen-dependent to FMN, and a carboxylic acid, according to ( cf. figure):

This reaction catalyzes a bacterial luciferase, a flavin-dependent monooxygenase. Since this simultaneously oxidized, the aldehyde to the carboxylic acid, is a mixed function oxidase. In all luminescent bacteria luciferase is a heterodimer of 76 ± 4 kDa size. It is composed of an α - and β - protein subunit (40-42 kDa and 37-39 kDa), which show little activity separately. The catalytic center is probably in the α - subunit. The luciferase ( phosphorerum Photobacterium, V. fischeri ) at pH values ​​between 6.0-8.5 and 6.0-9.5 ( Benecká harveyi ) is active, but not at temperatures above 30-35 ° C. A crystal structure of luciferase from Vibrio harveyi was solved with a resolution of 150 pm.

FMNH2 in free solution is unstable and easily oxidized. Bound enzyme but increases its stability and is attacked by nucleophilic oxygen at position C4a. The result is a 4a- hydroperoxide, which is present in unusually stable. This reacts with the aldehyde to a Peroxyhemiacetal which eventually decomposes to fatty acid and a 4 - Hydroxyflavin. The latter is in an excited state and falls back under light emission to the ground state. Thus the 4 - Hydroxyflavin is the actual light emitter. In the ground state this is hydrolyzed to FMN.

In the reaction catalyzed by the luciferase reaction blue-green light is emitted, the in vitro an emission maximum at about lambda max = 490 nm has. In vivo, however, wavelength maxima were observed from 472 nm to 545 nm. The reason for this is the transfer of excitation energy to fluorescent proteins via FRET. It has identified two classes of fluorescent proteins: blaufluoreszierende Lumazinproteine ​​( lumps) with lumazine as a chromophore ( P. phosphoreum, P. fischeri ). Alternatively, the gelbfluoreszierenden proteins ( YFPs ) form the second class, which, as a chromophore FMN or riboflavin ( P. fischeri strain Y -1). With the lumps, the emission maximum of 490 nm shifts to 476 nm, at 484 nm after YFPs of 534 nm for an energy transfer in accordance with the FRET of the luciferin-luciferase complex must be bonded to the respective fluorescent proteins. The quantum yield is 0.10 to 0.16.

The FMNH2 for the bioluminescent reaction is obtained by a riboflavin kinase with ATP consumption of riboflavin (vitamin B2). After the reaction, but FMNH2 regenerated from FMN, which catalyzes a flavin reductase NAD ( P) H consumption. Since the amount of aldehydes occurring in the bacterial cell ( see table above ) is sufficient only for a very short bioluminescence, the aldehydes are constantly regenerated. Here, the long-chain aldehyde is recovered from the fatty acid formed in the reaction, which is catalyzed in the so-called fatty acid reductase complex with consumption of ATP and NAD (P) H.

The bioluminescence reaction consumes a lot of energy because required for the regeneration of the components already two molecules of NAD (P ) H and one molecule of ATP. As a result, this light reaction must be controlled accordingly. In addition Flavinreduktasen have a higher turnover number than the luciferase. In an uncontrolled activity is producing too much FMNH2. By the rapid oxidation (see above) would thus much NAD (P) H to be wasted. This emphasizes the need for regulation.

Lux genes

All proteins that have something to do with the bioluminescent reaction be called lux genes encoding (Latin lux light). The subunits of luciferase encoded by the luxA or luxB genes, said luxB gene probably originated by gene duplication from the luxA gene. LuxA and LuxB have been successfully cloned as a marker. LuxC, D and E coding for the Fettsäurereduktasekomplex.

Coelenterazine, the common chemical component of many bioluminescent marine organisms

The work of Milton J. Cormier at the Seefedernart Renilla reniformis and Frank H. Johnson at the jellyfish A. victoria the luciferin coelenterazine was discovered. This is very prevalent among bioluminescent marine organisms, such as representatives of the Cnidarians ( Cnidaria ) ctenophores ( Ctenophora ), molluscs ( Molusca ), arthropods ( Atropoda ) and chordates ( Chordata ). Coelenteratzine but was not discovered in terrestrial life forms or segmented worms ( Annelida ). Sometimes they also occur in small amounts in non- bioluminescent organisms, such as in fire sponge Microcina prolifera. This also does not contain luciferase.

Coelenetrazin has a Aminopyrazingrundstruktur and is as light- emitting component as a bona fide luciferin ago. Often, it is also bound as a chromophore in Photo proteins such as aequorin, obelin or Symplectin. Similarly, derivatives of coelenterazine used by many marine organisms.

Modified coelenterazine is hardly stable in neutral aqueous solutions, there is easily oxidized by atmospheric oxygen. In methanol there exists stable. There fluoresces yellowish ( ε = 9800 M-1 · cm -1, lambda max = 435 nm). General coelenterazines react with oxygen to Coelenteramiden. Here enters a decarboxylation, it forms the anion of a Coelenteramides. This is the light emitter, so that in general, blue light is emitted. This reaction can be biokatalysiert ( bioluminescence ), but also takes place spontaneously ( chemiluminescence ). The bioluminescent reaction is explained in the following paragraph.

Mechanism

In 1962, the photoprotein aequorin was isolated from Aequorea victoria, and identified 1974 coelenterazine as the luciferin. How, then run the biochemical mechanisms for the luciferin - luciferase system with Imidazolpryazinen, was shown in 2000 on the basis of A. victoria. Here, the aequorin plays an essential role. Aequorin is a photoprotein small and located on the edge of the screen in the jellyfish. In aequorin luciferin ( coelenterazine ) is already connected with a peroxide bridge with the protein part. As a result, the photoprotein already leads the oxidizing agent is O2. In enzyme-linked form of coelenterazine may as well be kept for a long time. Aequorin has three binding sites for calcium ions. When calcium ions bind to the conformation of the protein changes in such a manner that intramolecular reaction with the coelenterazine is triggered. It reacts initially unstable Dioxetanonring so that after removal of CO2 is produced finally, the anion of coelenteramide. After relaxation to the ground state of a photon with a wavelength of lambda max = 465 nm is emitted. Because of this blue glow the protein as the blaufluoreszierende protein (blue fluorescent protein) ( BFP) is called. The photoprotein is finally regenerated in the presence of coelenterazine and molecular oxygen.

Aequorea victoria fluoresces but not blue, but green. The reason is that the blue fluorescent protein transfers the energy of the bioluminescence reaction radiationless on the so-called green fluorescent protein ( GFP).

Watasenia - luciferin

The bioluminescent deep-sea squid Watasenia scintillans was first described in 1905 (then known as Abraliopsis scintillans ). He has numerous Photophore the entire body, the bluish glow like a starry sky. For the bioluminescence reaction, a modified coelenterazine is necessary. This is one of coelenterazine disulfate and 1976 was isolated from the liver of the squid. It is called Watasenia - luciferin. In neutral aqueous solutions, it is unstable and tends to auto-oxidation ( chemiluminescence ), which is induced in particular by the presence of hydrogen peroxide and iron ( II) ions. In aqueous solutions, the luciferin is strongly fluorescent ( lambda max = 400 nm).

The Watasenia - luciferin is converted by a not yet isolated, membrane-bound luciferase, is emitted in consequence of blue light ( lambda max = 470 nm). The reaction has a pH optimum of 8.8 and required in addition to molecular oxygen ATP and Mg2 . For the reaction mechanism has been proposed that by means of the luciferin, ATP is adenylated so that it can bind to the luciferase. The further course of the reaction involves the formation of a Dioxetanonringes and finally the Coelenteramidanions, as generally shown above. The result is this light between 400-580 nm ( lambda max = 470 nm).

Vargula luciferin

The ostracods of the kind Vargula hilgendorfii (also referred to as Cypridina hilgendorfii ) secrete a luminescent liquid out into the sea water, if they feel threatened. Biochemical studies of the luciferase -luciferin system contained in these ostracods were launched at the beginning of the 20th century by Harvey. Meanwhile, it is examined closely.

The luciferin, Vargulin, was isolated in 1957 and identified in 1966 as Imidazolpryazinkomponente. It is soluble in water, methanol and alcoholic solvents. Vargulin has a yellow color in neutral solutions in methanol shows a maximum absorption at lambda max = 432 nm with a molar extinction coefficient of ε = 9000 M-1 · cm -1. In aqueous solutions, it is also slightly fluorescent ( excitation maximum at lambda max = 540 nm). Vargulin is very unstable and is especially by atmospheric oxygen, but also lead (IV ) oxide, oxidized. It can also light emitted, so here - especially in organic solvents such as diglyme - a chemiluminescent reaction is present.

In the ostracods Vargulin is implemented through a luciferase to coelenteramide, the oxyluciferin, where blue light is released ( lambda max = 463 nm). The luciferase is 60-70 kDa monomer with 555 amino acids. It has many cysteines and is an acidic protein (isoelectric point of 4.35 ).

From the bioluminescent reaction Vargulin is bound to the luciferase and oxygenated at the C2 atom. The result is a peroxide anion, which cyclizes to Dioxetanonring. This spontaneously decarboxylates to form the anion of the Coelenteramids, which is in an excited state. After the emission of a photon of this falls back to the ground state, oxyluciferin is released. The light emitter of the reaction is the oxyluciferin linked to the luciferase. The quantum yield is temperature - and pH - dependent, and is for Q = 0.30. As a side reaction is produced from 10 to 15% also etioluciferin, thereby but no light is emitted.

1966 was suggested that luciferin that is composed of L-arginine, L-isoleucine and L-tryptophan. For this, there is now growing evidence.

Dehydrocoelenterazin from Symplectoteuthis oualaniensis

Symplectoteuthis oualaniensis ( Japanese name Tobi- ika) is a widely used in the Pacific and Indian Ocean squid. The first study of its bioluminescence was published in 1981. The squid is Dehydrocoelenterazin to by a special photo- protein, which is referred to as " Symplectin ". There is as with other photoproteins ( aequorin, obelin ) as the chromophore via a cysteine ​​before covalently bound. The emitted light is bluish in the implementation, different emission maxima were given lambda max (456 nm, 470 nm, 480 nm). Bound Dehydrocoelenterazin is oxygenated at the C2 position, after Biolumenszenzreaktion arises coelenteramide and apo - " Symplectin ". The latter is regenerated by a molecule Dehydrocoelenterazin back to " Symplectin ".

The closely related octopus Symplectoteuthis luminosa ( Japanese name Suji - ika) also shows a bioluminescence. The involved chemical components and the mechanism of the bioluminescence reaction are similar or identical to those of S. oualaniensis. From the liver of the squid larger amounts of Dehydrocoelenterazin can be isolated.

Non-classical luciferin-luciferase systems

The Latia - luciferin

The case of New Zealand freshwater snail ( Latia neritoides ) occurring luciferin is a terpenoid aldehyde and is termed Latia - luciferin. The luciferin is highly hydrophobic, fat-soluble and a colorless liquid. Its absorption maximum is at lambda max = 207 nm, the molar extinction coefficient at 13,700 M -1. Since it is unstable, it can spontaneously hydrolyze to formic acid and an aldehyde. However, for the bioluminescence reaction of the latter is not active. If the enol formyl group is replaced with an enol - ether group, the luciferin is also no longer active.

The Latia - luciferin is catalytically converted to a ketone (oxy- luciferin ), which through a 173 kDa, colorless and non-fluorescent luciferase ( EC 1.14.99.21 ) is catalyzed. It is a Homohexamer, the individual subunits are approximately 30 kDa in size.

For the reaction is also a cofactor in addition to the luciferin, luciferase and oxygen needed, the 39 kDa protein purple ( purple -colored protein). This one is red fluorescent and seems to be a kind of activator for the bioluminescence reaction. It is for this but not absolutely necessary, since it can be replaced, for example by ascorbate and NADH. Even without purplish protein may run the bioluminescent reaction. In the reaction resulting from one molecule of luciferin, oxygen and water, respectively, an oxidized luciferin molecule and two molecules of formic acid:

Light is released, the emission maximum is at lambda max = 536 nm. Therefore, the mucus of the snail, which is secreted by mechanical stimuli, eg, light green to luminesce appears. The reaction efficiency of the bioluminescent reaction is very low, since the quantum yield Q is about 0.003 (25 ° C) and 0.0068 (8 ° C). To increase the quantum efficiency, can be added to the reaction, NADH (0.25 mM) and ascorbate (1 mM) so that it rises to 0.009 (25 ° C). However, in this case rise to other reaction products, which is represented by the following equation:

Whether a dioxetane ring is formed in the reactions as an intermediate, is still under discussion. The resulting oxy - luciferin but unlike oxy - luciferin firefly no fluorophore. It is believed that the energy released in this reaction energy is transferred to the actual emitter, a protein-bound flavin or a flavin - like group.

Luciferin from Diplocardia longa

The luciferin of the worm Diplocardia longa is a simple aldehyde, N- isovaleryl -3- aminopropanal. It is soluble in polar solvent ( methanol, ethanol, acetone, methyl acetate ), but not in non-polar solvents such as hexane, or carbon tetrachloride. The special feature of the bioluminescent reaction is the fact that hydrogen peroxide is required in lieu of molecular oxygen. The corresponding luciferase, an approximately 300 kDa, strongly asymmetric enzyme then converts the activated form, a peroxide adduct to. The luciferase likely to need copper, it is emitted blue-green light ( l max = 507 nm). However, you do not know what purpose can have bioluminescence in worms generally. Again, the actual emitter of the bioluminescence reaction has yet to be identified.

The quantum yield of this reaction is Q = 0.002 is very low.

Applications

Diagnostics

By means of the luciferin-luciferase system luminescent beetles, the presence of ATP can be checked quickly in the samples. This is utilized, for example, in the food industry in order to detect bacterial contamination. ATP only occurs in living organisms, which can be detected in food by the bioluminescence reaction.

Because the light is dependent aequorin reaction of calcium ions, the concentration of calcium ions can be measured by this system. The first application dated to 1967, when it was detected by means of aequorin, intracellular changes in calcium concentrations in muscle cells. After cloning of aequorin in bacteria could measure the calcium concentration bacterial cytosol. Moreover, it is possible to clone the aequorin into eukaryotic cells. Then one could measure the change in cytosolic calcium concentration after contacting the plant, or by a cold-shock for example in transgenic plants.

Genetic Engineering / Biotechnology

Luciferases are often used in molecular biology as a marker: organisms that have received the gene and incorporated into their genome, light when the supply of luciferin. So you could, for example, demonstrate whether genes that you would like to bring in organisms are actually expressed. It is simply the gene to be expressed is coupled to the a gene encoding a luciferase. By such a reporter gene may also be identified in the promoter regions of the genome. The luciferase genes of Photinus pyralis and Renilla reniformis usually be used commercially. In this case, both enzymes are often the same approach to the use ( " dual- luciferase assay" ).

Moreover, it is possible to measure by light reaction protein -protein interactions, signals in signal transduction processes, and the activity of cellular receptors.

For live animal model ( bioimaging ) the use of Luciferasereportern was also established. In the field of cancer research can be tracked using markers tumor growth or metastasis. In addition to living animals, the protein expression may be visualized by luciferase-luciferin systems.