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Bio- and Chemiluminescence, Part II

Dateline: 03/16/98

By Alan Bruzel

Bio- and Chemiluminescence, Part I defined bioluminescence and chemiluminescence as processes generating unstable molecular structures, and fluorescence and phosphorescence as processes generating unstable electronic structures. This article explores some bio- and chemiluminescent mechanisms and applications.

Bioluminescence

Most bioluminescent phenomena take place in marine organisms and may change the color of large areas of the ocean. Macbeth, after committing regicide, proposed an alternative mechanism:

No, this my hand will rather
The multitudinous seas incarnadine,
Making the green one red.
Macbeth Act II, Scene II

Ready administration of the ferruginous catalysts (keenly honed) provides dramatic interest, but those chemistries more sparing of laboratory staff and equipment compel our attention. Additionally, using cloned light-emitting systems engenders fewer guilt feelings than squeezing the gene products from natural sources.

The general reaction pathway for bioluminescence involves synthesis of an unstable intermediate, followed by its breakdown and visible light emission.

substrate + enzymeunstable intermediate

unstable intermediatestable end product + light

Each enzyme and substrate combination (a substrate is the molecule acted upon by the enzyme) produces a wavelength of light unique to the organism, as in the following examples:

Bacteria:
In Vibrio fischeri the substrate is a long chain aliphatic aldehyde, the enzyme is bacterial luciferase, and the wavelength of the emitted light is 495 nanometers (nm) (blue-green). There is a curious sidelight to this story. The squid Euprymna scolopes accumulates Vibrio fischeri in its light organ, attaining a concentration of 10¹⁰ to 10¹³ bacterial cells per milliliter. At this concentration, the bacterial luciferase gene complex continuously manufactures both luciferase and its autoinducer, resulting in a steady source of light. The squid provides food and protection for the bacteria, and the bacteria provide the means of diffusing the squid's shadow, making it a less likely target for potential predators.

Jellyfish:
In Aequorea victoria the substrate is coelenterazine (an imidazopyrazinone), the enzyme is aequorin, and the wavelength of the emitted light is 469 nm (blue). There is another interesting story here as Aequorea demonstrates both bioluminescence (from the aequorin/coelenterazine complex) and fluorescence (mediated by its green fluorescent protein). The 469 nm blue light from the bioluminescent reaction activates Aequorea's green fluorescent protein to fluoresce at 509 nm (green).

Insect:
In Photinus pyralis the substrate is the bisthiazole, luciferin, the enzyme is firefly luciferase, and the wavelength of the emitted light is 562 nm (yellow-green). Photinus, commonly called the firefly or lightning bug, is a beetle (order Coleoptera). It is neither a fly (order Diptera) nor a true bug (order Hemiptera).

The inexactitude of these common names must fill the entomologist with horror. Butterflies (Lepidoptera), caddisflies (Trichoptera), damselflies and dragonflies (Odonata), dobsonflies and snakeflies (Neuroptera), mayflies (Ephemeroptera), scorpionflies (Mecoptera), and stoneflies (Plecoptera) all have two pairs of wings. Dipterans possess but one pair. Perhaps it was a poet who attached the cognomen "fly" to so many disparate flying insects. In Samuel Johnson's The History of Rasselas, Prince of Abissinia, Chapter X, Imlac defines the poet, not the naturalist, as one who "does not number the streaks of the tulip." Johnson knew better, affirming to Boswell on April 18, 1783, that "Counting... brings everything to a certainty, which before floated in the mind indefinitely."

Bioluminescence provides numbers for the food industry by quantifying degrees of cleanliness. The analyst puts a wipe sample from a counter top into a solution of luciferin and luciferase. The luciferin/luciferase system requires the presence of adenosine 5'-triphosphate (ATP) for its bioluminescent activity. Because food residue and bacteria contain ATP, the amount of light emitted by the reaction is proportional to the sample's contamination.

Chemiluminescence

Unlike bioluminescence, chemiluminescence does not restrict itself to emitting visible light only. Nitrogen monoxide (NO) combines with ozone (O₃) to form the unstable intermediate, activated nitrogen dioxide (NO₂^*). This molecule returns to the de-excited nitrogen dioxide state (NO₂) with the emission of invisible 1200 nm light (near infrared).

NO + O3NO2* + O2

NO2*NO2 + light

This reaction can test for the presence of ozone (see this site's article Chlorofluorocarbons and Ozone) or nitrogen monoxide. However, most ozone measurements use ozone's ultraviolet light absorption, not its reaction with nitrogen monoxide. For nitrogen oxide analysis, a detector with a built-in ozone generator is available.

Of the many experimental techniques using chemiluminescence is the measurement of hydrogen peroxide in air. Detection levels range from 23 parts per trillion to 3.37 parts per billion, underscoring the potential of chemiluminescent reactions in measuring extremely low concentrations of suitable reactants.

Hydrogen peroxide, albeit at much higher concentrations, also finds use as the oxidizing agent in the familiar "lightstick." As in Aequorea, this is a collaborative effort of chemiluminescence and fluorescence. Bending a new lightstick breaks an enclosed glass ampoule releasing hydrogen peroxide. The surrounding oxalic phthalate ester oxidizes to an unstable four membered ring intermediate. Chemiluminescence originating from the breakdown of this intermediate excites the dye 9,10-bis-(phenylethynyl)anthracene to green fluorescence.

A final example of chemiluminescence meanders close to the borderline of bioluminescence, but the substrate, phosphorylated 1,2-dioxetane, is man-made. The enzyme is alkaline phosphatase and the wavelength of the emitted light is 466 nm (blue).

Molecular biology routinely requires comparison of a known DNA fragment (one having a defined nucleotide sequence) to unknown DNA fragments. The same methodology matches the DNA of a crime scene to a suspect's DNA. To make use of the above chemistry, the known DNA is covalently bound to alkaline phosphatase. A panel of unknown DNAs, immobilized on a test strip, are immersed into a solution of this enzyme-linked DNA. The DNA-alkaline phosphatase complex hydrogen bonds only to complementary DNAs on the test strip. The strip is removed and treated with the phosphorylated 1,2-dioxetane substrate. Those unknown DNA samples hybridizing to the enzyme-linked DNA probe (and thus having structural homology to it) are identified by the chemiluminescent reaction catalyzed by the alkaline phosphatase. The light signal is detected by scintillation counter, luminometer, or photographic film.

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