Fluorescent proteins and bioluminescence
There are many living organisms that emit light, including the firefly, the jellyfish and certain bacteria. Some of them emit light by absorbing specific wavelengths light and some emit light by consuming energies stored within the body.
Fluorescent proteins emit fluorescence by absorbing the energy of light. Jellyfish, coral, sea anemones and certain bacteria have such proteins in their bodies. On the other hand, bioluminescence comes from a chemical reaction inside the body; examples include the firefly and the noctiluca. The light-emitting substance is either located inside the cells (the firefly and Noctiluca) or secreted outside (Cypridinacea). Although proteins are involved in these chemical reactions, the proteins themselves do not emit light directly.
Both fluorescent proteins and bioluminescence are widely used in medical and biological research. Thousands of materials have been collected from the field. While only small amounts of the proteins have been extracted in such studies, the proteins have been made available through chemical synthesis or genetic engineering. The genes of the GFP (Green Fluorescent Protein) and its variants are available commercially and are applied in the fields of cell biology and molecular biology.
Fluorescent proteins
- Green Fluorescent Proteins and its variants <BFP, CFP, GFP, YFP, and DsRed>
- Phycobiliproteins <B-Phycoerythrin(B-PE), R-Phycoerythrin(R-PE), Allophycocyanin(APC)>
The GFP occurs naturally in jellyfish; amino acids in the 65th to 67th positions have been found to emit fluorescence by ultraviolet or blue light excitation (DsRed is found in the sea anemone). The GFP gene is used in research to visualize or track activity at the cellular level through its expression in cells.
The phycobiliproteins are obtained from bacteria and algae. These proteins contain a covalently linked fluorescence group. Specimens labeled with phycobiliproteins are used mainly for flow cytometry and can also be observed in microscopy.
| Fluorescent Proteins | Excitation Wavelength/nm | Emission Wavelength/nm |
|---|---|---|
| GFP (Wild Type) | 395/475 | 510 |
| ECFP | 433 | 475 |
| EGFP | 488 | 507 |
| EYFP | 513 | 527 |
| DsRed | 558 | 583 |
| B-PE | 545/565 | 575 |
| R-PE | 480/545/565 | 578 |
| APC | 650 | 660 |
Bioluminescent proteins
- Aequorin
- Luciferin-Luciferase
Among the bioluminescent proteins, these are the two types most frequently used in biological research. Aequorin is a bioluminescent protein complex isolated from the jellyfish and used in calcium ion concentration measurement. It includes the protein apoaequorin and a latently luminescent molecule. When a calcium ion binds to aequorin, the complex breaks down into apoaequorin and a luminescent molecule, which emits blue light (wavelength ~ 466 nm). Unlike measurements using the fluorescent dye probes, calcium ion concentration measurement using aequorin does not require excitation and is not affected by autofluorescence. Synthetic apoaequorin is available at present. The apoaequorin gene can also be used in calcium ion concentration measurement through its expression in mitochondria.
Luciferin is the bioluminescent molecule obtained from the firefly; it emits luminescent light when it is oxidized by an enzyme (protein) named luciferase (wavelength up to 560 nm). As ATP is indispensable in the Luciferin-Luciferase reaction of the firefly, this reaction is used for an ATP detection kit with very high sensitivity. Such ATP detection kits are available commercially. The luciferase gene has also been obtained and can be expressed inside cells.
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