Biosulem Essay, Research Paper

The instance of bioluminescence is both mysterious and relatively common. Most of us have observed a meandering firefly on a hazy summer evening. Few of us have understood what processes were going on in the firefly abdomen that resulted in the emission of light by a living organism. The phenomenon of bioluminescence is not limited to fireflies and glowworms. In fact, bioluminescence affects many different types of species. It helps them survive and can be emitted through many different means.

This paper will explore instances of bioluminescence, its diversity and its evolution. In addition, it will expand on history of the study of bioluminescence, its chemical mechanisms and the actual release of light. The purpose of this paper is to review important aspects of bioluminescence, as well as to generate interest in this fascinating subject.

The phenomenon of bioluminescence can be observed in many different settings. Animals that emit light include bacteria, dinoflagellates, fungi, fish, insects, shrimp and squid. Bacteria are the most abundant of bioluminescent species. Most free-living species live in the ocean (Meighen, 1991). Scientist Peter Herring has worked extensively with marine bioluminescence. In the water, bioluminescent tissues can be point source emitters or external glandular organs of bacterial symbionts (Herring, 1985). External glandular organs, where a luminous secretion is released from an opening in the shell, can be found on crustaceans. Symbiont glands can be found on fish or cephalopods. The luminescent bacteria harbored in the gland will produce light for the host. Anglerfish have been known to expel their symbionts in a luminous cloud. As for luminous tissue directly in an organism, positioning on the periphery of the animal is common. Luminous tissue such as photocytes can be sighted on dorsal and ventral areas and down the fin rays (Herring, 1985). For example, marine annelids named polynoid scale worms have photocytes for ventral epithelial cells. They are unique in that their scales flash rapidly when they are attacked. When escaping danger, scale worms sometimes leave behind their luminescent scales as a decoy (Wilson and Hastings, 1998). In cephalopods, photophores are on the tips of tentacles (Herring, 1977). Another placement of photophores is in the eye. In certain gonostomatid and myctophid fishes, the photophores shine into the eye. Almost any tissue can be modified to luminesce (Herring, 1985).

Bioluminescence is not limited to glowworms and fireflies. Of all the phyla in the plant and animal kingdoms, 50% of them include luminescent organisms (Harveyi, 1952). These organisms live on land, in freshwater and in the sea (Meighen, 1991). The diversity of luminescent organisms in their enzymes and substrates suggests that bioluminescence evolved many times independently. In fact, 30 different systems exist today (Hastings, 1983, Rees et al, 1998, Wilson and Hastings, 1998). The ability to emit light has helped species survive and reproduce throughout the ages. It can aid survival in the dark, in the deep sea or at night (Herring, 1985). In bacteria and fungi, bioluminescence evolved with the cell respiratory mechanism (Hastings, 1983). The diverse nature of bioluminescent substrates and enzymes, luciferins and luciferases, produce further argument for evolutional diversity (Hastings, 1983, Hastings, 1996, Rees et al, 1998).

Part of the reason that bioluminescence has evolved and flourished is that it has many beneficial uses to the organism. Squid use light emission as a decoy, jellyfish flash and dinoflagellates glow as an alarm. These three animals luminesce to avoid predators. Anglerfish activate photophores to lure in smaller fish. Squid and fish luminesce to camouflage themselves. Flashlight fish emit light to aid their vision. These organisms benefit from bioluminescence in their hunt for prey (Hastings, 1983). Fireflies benefit from their glow during courtship (Hastings, 1983).

Bioluminescent systems exhibit diversity not only in types of chemicals produced and reasons for producing them, but in methods of controlling light emission. Bacteria glow continuously because their luminescence is a shunt of the respiratory pathway (Hastings, 1983). Fish and squid with symbiont light organs control light emission with a shutter or with a secondary cellular control (Hastings, 1983). Flashing is achieved by photic stimulus in the luminous organs (Buchner, 1965). In turn, this stimulus could be controlled by oxygen or a peroxide intermediate. The degree of luminescence can also be controlled by the cellular enzyme content, which varies upon conditions and gene expression (Hastings, 1983). Fireflies control their biochemical reactions during the reaction of luciferin with ATP. This forms a luciferyl-AMP, the substrate that reacts with oxygen to emit light (Hastings, 1983). A unique method of light reaction control can be found in Gonyaulax (a dinoflagellate). pH is the dominant variable for reaction control in this system. The luciferin binding protein, a substrate bound to a protein, releases the luciferin when the pH moves from 8 to 6 (Hastings, 1983, Lee et al, 1993). It is this controlled pH shift that facilitates flash emission in Gonyaulax.

The study of bioluminescence has been going on for centuries. One of the earliest advances came from Boyle s work in the mid-1600 s. In 1667, Boyle observed that a piece of wood glowed in the presence of air, but dimmed when he pumped the air out of the system. Boyle wrote the following:

a piece of shining wood gave a vivid light which was manifestly lessened

(at) about the seventh suck (of air), losing its light more and more as the air was

still further pumped out Wherefore we let in the outward air and had the pleasure

to see the seemingly extinguished light revive so fast and perfectly, that it looked

to us almost like a little flash of lightning

We now know that the flash was a product of the reaction of O2 with a reduced enzyme intermediate (Hastings, 1983). However, early scientists thought of bioluminescence as a problem. The phenomenon did not seem to fit into known evolutionary patterns. Charles Darwin wrote briefly about bioluminescence in Origin of Species under a section called Special difficulties of the theory of natural selection (Darwin, 1859).

One of the first modern scientists concerned with bioluminescence was Harvey. Harvey was confused with the sporadic distribution of the ability to luminesce throughout phyla (Hastings, 1983). He was truly ahead of his time when he argued the mechanisms of light production (Hastings, 1983). Harvey wrote in 1932:

in the simplest organisms, the bacteria and the fungi, light production

evolved in connection with the cell respiratory mechanism, by the devel-

opment of one of the hydrogen acceptors whose oxidation gives sufficient

energy to excite a compound similar to luciferase

Harvey s work further established the broadness of the oxygen requirement. He also found exceptions to the oxygen-requiring norm (Hastings, 1983).

Because bioluminescence has evolved independently on many occasions, it is difficult to provide a uniform description of how all bioluminescent organisms emit light. The emission of light comes from photons ( 50 kcal/mol) that are the result of exergonic reactions of molecular oxygen with varying substrates (luciferins) and enzymes (luciferases) (Baldwin et al, 1995, Hastings, 1975, 1983,1996, Meighen, 1991, Rees et al, 1998, Shimomura, 1975, Wilson and Hastings, 1998). The amino acid sequence of the luciferase will affect the color of light produced. Luciferin molecules can affect the colors of emitted light also (Hastings, 1983). Accessory proteins, such as green fluorescent protein, or GFP, can also manipulate the color or wavelength (Wilson and Hastings, 1998). The exciting aspect of the chemical process of bioluminescence is the excited state necessary for emission. This excited state is the dot on the exclamation point of the chemical process. The resulting excited-state molecule holds its bundle of sudden energy for a fraction of a second. When the electronically excited molecule cannot retain its energy any longer, it will release the energy in a package called a photon (Wilson and Hastings, 1998). This extremely short final step is what we observe when fireflies release light.

Each organism has its own bioluminescent style. In bacteria, the luciferase enzyme catalyzes oxidation of an aldehyde chain and a mononucleotide (Baldwin and Zeigler, 1992). Afterwards, the mononucleotide is bound to the enzyme. The luceferase-bound product reacts to yield a luciferase-bound flavin hydroperoxide (Wilson and Hastings, 1998). The next step involves the reaction of an aldehyde to form a (not yet isolated) postulated peroxyhemiacetal (E*FOOA). An enzyme-bound 4-alpha-hydroxyflavin is the emitter. The result of this complicated process is a quantum yield of 0.3 hv per reacting mononucleotide (Wilson and Hastings, 1998).

In the firefly, luciferin reacts with oxygen in the presence of the luciferase enzyme (Koo, Schmidt and Schuster, 1978). A high-energy dioxetanone molecule is produced (White et al, 1975). Afterwards, the dioxetanone loses CO2 and becomes electronically excited. In nature, the excited singlet state is formed with nearly 100% efficiency (Koo, Schmidt and Schuster, 1978). This truly amazing efficiency of nature allows fireflies to get more out of each abdominal flash.

Four proteins are involved in the bioluminescence of the sea pansy Renilla (Cormier, 1978). One of them is a sufolkinase which removes a sulfate from coelenterazine (Wilson and Hastings, 1998). Coelenterazine is the luciferin present in bioluminescent coelenterates. It binds to a luciferin binding protein, or LBP (Wilson and Hastings, 1998, Shimomura and Johnson, 1975, Anderson et al, 1974). Next, it is released from the protein, in the presence of calcium. Calcium is important because the LBP contains three Ca2+ binding sites (Wilson and Hastings, 1998). The oxidation of the released coelenterazine forms a dioxetanone intermediate, much like that of the firefly process. Once again, the dioxetanone breaks down, forming CO2 and an excited state oxidized luciferin (Koo, Schmidt and Schuster, 1978).

In the jellyfish Aequorea, oxygenated coelenterazine binds to a luceferase reaction intermediate. This intermediate is a photoprotein (Wilson and Hastings, 1998). The photoprotein has three calcium binding sites. The presence of calcium enables the completion of the reaction by triggering the excited flash (Wilson and Hastings, 1998). The bioluminescence of the Aequorea is green due to an accessory protein: green fluorescent protein. The GFP acts as a secondary emitter (Chalfie, 1995, Wilson and Hastings, 1998).

Now that we are familiar with the chemical process of bioluminescence, we come to the question, What exactly creates the excited state? . Through research, two different means have been identified (Faulkner, 1978). First, electron transfer can induce an excited state. Electron transfer is self-descriptive. One electron is transferred from a radical anion to a radical cation. This leaves the cation in the excited state. Studies of the excited state were conducted by JW Hastings in 1983. He found the following: This system wastes little energy, increasing the overall efficiency of the system, because the structures of the reaction participants are not changed. The second path to an excited state is more common and was discovered by Kopecky and Mumford in 1969. This system is based on four-membered ring peroxides. These peroxides, when heated, yield high amounts of excited products. Breaking down the compound can release about 100 kcal because the peroxide s O-O bond is weak and has gained energy from the earlier formation of two carbonyl double bonds. The ring peroxide mechanism is present in fireflies and cyprindia. The reaction involves the uptake of an O2 and the production of a CO2 from another carbonyl (Hastings, 1983).

Earlier in this article, green fluorescent protein was mentioned. GPF is one of several accessory proteins. These accessory photophores change the color of luminescence of the system. GPF is unique because the chromophore is covalently bound (Wilson and Hastings, 1998). Wilson and Hastings wrote:

The biosynthesis of the chromophore can be looked upon as requiring successive steps: proper folding of the peptide chain so as to bring residues 65 and 67 to the geometry appropriate for cyclization, dehydration, and finally oxidation to form a C=C double bond on the phenolic side chain of tyrosine and thus create the 4-hydroxycinnamyl part of the chromophore.

GFP will only fluoresce with the addition of oxygen. Its structure makes it thermally stable and resistant to proteolysis. In short, the characteristics of green fluorescent protein change the color of emitted light (Wilson and Hastings, 1998).

A second accessory protein is YFP or the yellow fluorescent protein. Because its chromopohores are not covalently bound, YFP changes the kinetics of the chemical reaction as well as the color of emitted light (Wilson and Hastings, 1998). One instance of YFP in action is the emission of yellow light from the bacterium Vibrio fischeri (Daubner et al, 1987). Its luminescence appears yellow because the YFP speeds up the decay of luciferase and destabilizes the enzyme-bound intermediate, which would be the peroxyhemiacetal in this case (Wilson and Hastings, 1998). YFP is strongest at 4*C and virtually non-existant at 20*C. As stated earlier the yellow emission originates from the complex, via an intra-complex energy transfer process (Wilson and Hastings, 1998).

Bioluminescence can be described as the emission of light by a living organism through chemical means. Bioluminescent organism include, but are not limited to, bacteria, dinoflagellates, fungi, fish, insects, shrimp and squid. Luminescent cells can be found anywhere on the body of a luminescent organism. This luminescence has evolved independently many times. The phenomenon of light emission has been studied for many years. It piqued the minds of scientists such as Boyle, Darwin and Harvey. The diversity of bioluminescence can be observed by studying the substrates and enzymes that are responsible for glowing. Bioluminescent substrates and enzymes are known as luciferins and luciferases. They (in most instances) react with molecular oxygen to create an excited state molecule which, in turn, releases a package of luminous energy called a photon. The excited state molecule releases this photon by means of electron transfer and is extremely efficient. Accessory proteins such as GFP and YFP affect the wavelength of emitted light (shifting it to green or yellow) by interacting with reaction intermediates. Bioluminescence is a useful and still-studied occurrence that, as simple and attractive as it seems, brings together many complex molecular reactions to achieve the emission of light by a living organism.