Bioluminescence

Bioluminescence is the production of light by living systems. The best-known example of this phenomenon is the characteristic glow of the firefly, but other luminous species include bacteria, fungi and other animals such as jellyfish, scale-worms, deep-sea squid, prawn and fish. In animals bioluminescence is used as a diversionary tactic when disturbed, to attract prey and of course as a mating signal during courtship. 

Bioluminescence is defined as the emission of light, without heat, by living organisms. It occurs in fireflies, glowworms, and some varieties of bacteria, fungi and deep-sea fish. The luminescence arises in the course of oxidation of compounds called luciferins with enzymes called luciferases. The composition of a luciferin is species dependent, but the oxidations can often be effected in quite low oxygen concentrations. 

The cold luminescence of the firefly arises from a cycle of reactions involving ATP, luciferin and the enzyme luciferase, all of which have been isolated. Luciferin reacts with ATP to form luciferin adenosine monophosphate. This is then oxidised by air to oxyluciferin with an anission of energy in the form of visible light (11.107). Luciferin is then regenerated by other processes. Luciferin contains an asymmetric C atom and only the D form produces light emission. 

ATP bioluminescence has become an important analytical tool. This luminescence can be utilised for a speedy and sensitive test for the number of bacterial cells present in bio samples. The ATP is extracted from the cells and assayed with firefly luciferase to produce luminescence which is proportional to the amount of ATP present [24]. Microorganisms can be detected and measured in soap, cosmetics and toothpaste [24a]. 

Bioluminescence is the process by which a molecule in the excited state emits light, which is then measured (Hartman et al., 1992). This process can be used to measure the amount of ATP produced by microorganisms as part of their metabolism. In theory, measurement of this compound should provide an estimate of viable cell numbers because higher populations of microorganisms produce more ATP. Because a single yeast cell will have generally more ATP than a bacterial cell, the detection limit for yeast could be as low as 10 cells (Hartman et al., 1992). These authors further suggested that a practical limit for bacteria is closer to 1000 to 10,000 cells. 

The luciferin-luciferase assay commonly used to measure ATP is as follows  

Bioluminescence is associated with the emission of light by living organisms such as the firefly, the glow-worm and various aquatic organisms. The production of light arises from a wide variety of sequences of biochemical reactions. A simplified explanation is that an enzymatic reaction is catalysed by luciferase and liberates a compound 

The radiation emitted by oxyluciferin (hv) is similar to the fluorescence that can be produced by irradiating oxyluciferin via the usual method [216]. 

Luciferin includes a whole family of compounds whose heterocyclic structures vary fix m one organism to another. Most luciferase enzymes use the same cofactors as metabolic processes (ATP, FMN, NADH), and bioluminescence is easily associated with other types of Inological reactions for analytical applications. Firefly ludferase is used tt follow processes that use adenosine triphosphate (ATP) as a cofactor, for example, the measurement of biomass, the detection of a bacterial infection, antibiotic assays, and the monitoring of other enzymatic reactions that consume or produce ATP. Luciferase catalyses all these reactions according to the following overall reaction scheme  

The radiation is emitted at A. = 562 nm, and the excellent quantum yield (88 %) makes the technique very sensitive. The detection level for creatin kinase in the diagnosis of myocardial infarction is of the order of a femtomole (fmol = lO-i mol). This enzyme is determined via the detection of ATP from the two following reactions  

Bacterial luciferase can be easily obtained from the culture of 

Chemiluminescence and Bioluminescence.—The function of thiazole and benzothiazole derivatives in the processes of bioluminescence and chemiluminescence continues to form the subject of significant investigations. Extensive contributions have been provided by White et al., ° who have reviewed this interesting and active field. They have shown that the luminescence of firefly luciferins involves intermediates having dioxetan structures. 

Firefly luciferin (111 R = R = H, X = OH) and a number of its analogues were synthesized by previously established reaction sequences from a variety of substituted 2-cyanobenzothiazoles (109) and cysteines (110). Amongst others, homoluciferin (112) and 5, 7 -dimethyl-luciferin (113) are accessible in this way. Their adenylates were produced by the condensation of the free acids with adenosine monophosphate in the presence of dicyclohexylcarbodi-imide. These were used in detailed spec-trophotometric studies of their chemiluminescence (initiated by bases), their fluorescence, and their bioluminescence under the influence of luciferase the possible mechanisms of these processes were discussed.  

The suggested mechanism for the red light emission by firefly luciferyl 

Yamamoto, S. Nakamura, K. Yoshimura, M. Yuge, S. Morosawa, and A. Yokoo, Bull. Chem. Soc. Japan, 1973, 40, 1509. 

See also M. J. Cormier, J. E. Wampler, and K. Hori, in Progress in the Chemistry of Organic Natural Products , Springer, Wien and New York, 1973, Vol. 30, p. 1. 

The best known example of bioluminescence is that of the glow worms, but many other examples are found among molluscs, fishes and mushrooms. Bioluminescence is fairly common among marine life, especially in the deep waters where sunlight cannot penetrate. 

Berzelius then extended his development to represent compounds, for example, copper oxide was identified as CuO and zinc sulfide as ZnS. And, conforming to Proust s law and Dalton s theory, Berzelius added algebraic exponents (later to become subscripts) to his system of atomic symbols— for example, water was denoted as H2O and carbon dioxide as CO2. 

Even though his atomic symbols were introduced in 1814, it was quite a few years before Berzelius s symbols were adopted by the chemistry community. But once accepted, they became the new international language of chemistry. 

Berzelius published more than 250 papers in his lifetime covering every aspect of chemistry. He was devoted to the entire field of chemistry, as can be seen by his efforts to bring order to the language of chemistry and to insist on quantitative excellence in all its areas. He died in 1848 and is buried in Stockholm, Sweden, see also Atoms Dalton, John. 

William H. (1993). The Norton History of Chemistry. New York W. W. Norton. 

Bernard (1976). Crucibles The Story of Chemistry from Ancient Alchemy to Nuclear Fission. New York Dover. 

The electrogenerated chemiluminescence (ECl) of five l-amino-3-anthryl-9-propane derivatives has been studied in tetrahydrofuran. Emission from intramolecular exciplexes in ECl spectra and weak emission from the locally excited anthracene moiety were observed. The influence of triplet state interaction in ECl emission is discussed. The chemiluminescent decomposition of three a-peroxy-lactones gives CO2 and the corresponding ketone in high yield. The chemiluminescent species produced has been investigated in some detail by measurements of lifetime, energy-transfer activation parameters, and photochemical reactions. 

The quenching of peroxidized luminol chemiluminescence by reduced pyridine nucleotides has been reported. Neither superoxide nor hydroxyl radical scavengers were found to quench the chemiluminescence of luminol in the presence of horseradish peroxidase and H2O2. Both chemi- and bio-luminescence of firefly luciferin have been investigated and a dioxetanone mechanism proposed for the light-producing pathway.  

The relation between structure and triboluminescence has been investigated in two polymorphic systems, viz, hexaphenylcarbodisphosphorane and anthranilic acid. ° Only one phase is triboluminescent. A correlation between triboluminescence and unit cell symmetry groups is given. 

The detection and possible applications of lyoluminescence are described in an interesting paper by Ettinger et It is shown that lyoluminescence can be used to measure the radical scavenging activity of chemical compounds, particularly those with potential for use as radioprotective agents. 

Adenosine triphosphate (ATP) is produced in the course of metabolism by all living cells and, upon cell death, theoretically rapidly degrades. Thus, measurement of the compound may provide an estimate of biomass (and, hopefully, viable cell numbers). ATP released from living cells after boiling the sample in Tris buffer is measured using the luciferin-luciferase assay in which reduced luciferin is oxidized in the presence of oxygen, luciferase enzyme, ATP, and Mg  

The light produced is measured by a luminometer and is directly proportional to ATP present in the sample. 

Dowhanick and Russel (1992) report that the method is capable of directly detecting 100 yeast cells per sample. To detect very low numbers ( 5/sample), it is necessary to incubate the sample for several hours at optimal temperature to allow for replication. 

Despite the deficiencies noted above, ATP-bioluminiscence technology is used in sanitation monitoring, where immediate results are required prior to beginning operation. Presently, there are several luminometers and test kits being marketed. Once the monitor is in place, the cost per test (which includes sample collection container, swab, and reagents) is near 3. 

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Later, fireflv oxyluciferin was successfully synthesi2ed (403. 408) and has been isolated and identified in firefly lanterns (luciola cruaciata) after the lanterns were treated with pyridine and acetic anhydride to prevent decomposition (409). In 1972, Suzuki and Goto firmly established that oxyluciferin is involved in the bioluminescence of firefly lanterns and in the chemiluminescence of firefly luciferin (403. 410).. A. mechanism involving a four-membered ring cyclic peroxide has been proposed for the reaction (406. 411). However, it was not confirmed by 0 -labelinE experiments (412). 

The release of a photon following thermal excitation is called emission, and that following the absorption of a photon is called photoluminescence. In chemiluminescence and bioluminescence, excitation results from a chemical or biochemical reaction, respectively. Spectroscopic methods based on photoluminescence are the subject of Section lOG, and atomic emission is covered in Section lOH. 

Bioluminescence can also be used as the basis for immunoassay. For example, bacterial luciferase has been used in a co-immobilized system to detect and quantify progesterone using a competitive immunoassay format (34), and other luciferase-based immunoassays have been used to quantify insulin, digoxin, biotin, and other clinically important analytes (35). 

L. J. Kricka, ed.. Analytical Applications of Bioluminescence and Chemiluminescence, Academic Press, Inc., New York, 1984. 

Long before 1,2-dioxetanones were isolated, they were proposed as key intermediates in bioluminescence (58—60). This idea led to the discovery of a number of new chemiluminescent reactions. For example, (23) reacts with to give (25). The hydroperoxide (24) has been isolated and is... 

X = Cl) was based independently on the dioxetanone (61) and concerted peroxide decomposition (6,8,62) theories. Possible examples of dioxetanones in bioluminescence are discussed later. 

Bioluminescence functions in mating (fireflies, the Bahama fireworm), in the search for prey (angler fish, Photmus fireflies), camouflage (hatchet fish, squid), schooling (euphausiid shrimp), and to aid deep water fish (flashlight fish, Photoblepharon to see in the dark ocean depths. 

The intensity of bioluminescence emission is > 2 x 10 photon /s-cm in the dinoflageUate Gonyaulax and the spectmm of light emission ranges from 450—490 nm (blue) in deep sea species, 490—520 nm (green) in coastal water species, and 510—580 nm (yeUow-green) in terrestrial and freshwater species. 

Firefly. Firefly luciferase (EC 1.13.12.7) is a homodimeric enzyme (62 kDa subunit) that has binding sites for firefly luciferin and Mg ATP . Amino acid sequence analysis has iadicated that beetle luciferases evolved from coen2yme A synthetase (206). Firefly bioluminescence is the most efficient bioluminescent reaction known, with Qc reported to be 88% (5), and at 562 nm (56). At low pH and ia the presence of certain metal ions (eg, Pb ", ... 

The carbonyl compound (43) has also been synthesi2ed, and its fluorescence spectmm has been shown to match the bioluminescence spectmm under equivalent conditions (214). The details of the excitation step are unclear and a dioxetanone mechanism (59,215) may apply to the reaction. 

Goelenterate. Coelenterates Penilla reformis (sea pansy) -cradViequoreaforskalea (jelly fish) produce bioluminescence by similar processes (223). The basic luciferin stmcture is (49) (224) and excited amide (50) is the emitter. The stmctural relationship to Varela is evident. A stmctural analogue where R = CH is active ia bioluminescence. The quantum yield is about 4% (223), with at 509 nm (56). This reaction iavolves a charge transfer between green fluorescent proteia and the excited-state coelenterate oxylucifetin. 

Genes lux) encoding luciferase and the other proteias iavolved ia the bioluminescent reaction have been cloned. PuxA. and luxP genes code for the luciferase subunits, and the fatty acid reductase complex is coded by the luxCDE genes. 

Latia. The freshwater snail Eatia has been reported to provide bioluminescence by the following reaction (236). 

Analytical Applications. Chemiluminescence and bioluminescence are useful in analysis for several reasons. (/) Modem low noise phototubes when properly instmmented can detect light fluxes as weak as 100 photons/s (1.7 x 10 eins/s). Thus luminescent reactions in which intensity depends on the concentration of a reactant of analytical interest can be used to determine attomole—2eptomole amounts (10 to 10 mol). This is especially useful for biochemical, trace metal, and pollution control analyses (93,260—266) (see Trace and residue analysis). (2) Light measurement is easily automated for routine measurements as, for example, in clinical analysis. 

Direct Metal Analyses. Calcium ion can be detected to a lower limit of 10 M hy Aequorea bioluminescence. Strontium interferes to a minor extent (270,271). 

Clinical Analysis. A wide range of clinically important substances can be detected and quantitated using chemiluminescence or bioluminescence methods. Coupled enzyme assay protocols permit the measurement of kinase, dehydrogenase, and oxidases or the substrates of these enzymes as exemplified by reactions of glucose, creatine phosphate, and bile acid in the following ... 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Bioluminescence in vitro chemosensitivity assays are now used to assess the sensitivity of tumor cells (obtained by surgical or needle biopsy) to different dmgs and combinations of dmgs. Cells are grown in microwell plates in the presence of the dmgs at various concentrations. If the tumor cells are sensitive to the dmg then they do not grow, hence total extracted cellular ATP, measured using the bioluminescence firefly luciferase reaction, is low. This method has been used to optimize therapy for different soHd tumors and for leukemias (306). 

A. Lundin, in A. Szalay, L. J. Kiicka, and P. Stanley, eds.. Bioluminescence and Chemiluminescence Status John Wdey, Sons, Ltd., Chichestei,... 

P. E. Stanley and L. J. Kricka eds.. Bioluminescence and Chemiluminescence Current Status, ]ohxi Wiley Sons, Chichester, U.K., 1991. 

Chemiluminescence. Chemiluminescence (262—265) is the emission of light duting an exothermic chemical reaction, generaUy as fluorescence. It often occurs ia oxidation processes, and enzyme-mediated bioluminescence has important analytical appHcations (241,262). Chemiluminescence analysis is highly specific and can reach ppb detection limits with relatively simple iastmmentation. Nitric oxide has been so analyzed from reaction with ozone (266—268), and ozone can be detected by the emission at 585 nm from reaction with ethylene. 

K. Van Dyke, ed.. Bioluminescence and Chemiluminescence Instruments and Applications, CRC Press, Boca Raton, Fla., 1985. 

Chemiluminescent labels, in which the luminescence is generated by a chemical oxidation step, and bioluminescent labels, where the energy for light emission is produced by an enzyme-substrate reaction, are additional labeling types (39,42). Luminol [521 -31 -3] CgHyN202, and acridine [260-94-6] C H N, derivatives are often used as chemiluminescent labels. 

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