Osamu Shimomura

Structure of the chromophore of Aequorea green fluorescent protein

In the bioluminescence of the jellyfish Aequorea, the green fluorescent protein (GFP) plays the role of the light emitter [ 11. The energy needed for the emission of light is produced in the Cap-triggered reaction of the photoprotein aequorin [Z] that coexists with GFP in the photogenic organ of the jellyfish. The energy is then transferred to GFP molecules by the Foster-type mechanism [ 11. The excited state of GFP thus formed ultimately dissipates the energy in the form of green light (X,, SO9 nm). In the absence of GFP, aequorin emits blue light (A,, 470 nm) [ 11. Bioluminescence of various other coelenterates apparently also involves similar green fluorescent proteins [3]. GFPof the sea pansy Renilla, in addition to Aequorea GFP, has been extensively studied [4]. It has been thought that the chromophores of all GFPs are the same [5], although until now information has not been reported concerning the chemical nature of the chromophore from any source. The structural knowledge concerning the chromophore of Aequoveu GFP reported in the present paper may help to identify the chromophore of other GFPs.

Mechanism of the luminescent oxidation of Cypridina luciferin

In an aqueous medium with Cypridina luciferase, or in an aprotic solvent without luciferase, the aerobic oxidation of Cypridina luciferin is accompanied by emission of light (1,2,3). The products of the luminescent oxidation have been reported as oxyluciferin and CO2 (4,5,6), although the oxyluciferin can be further converted to etioluciferin by acid or slowly by luciferase (4,7).

Widespread occurrence of coelenterazine in marine bioluminescence

Abstract 1. 1. Coelenterazine is a compound having a key role in the light-emitting process of bioluminescent coelenterates. Present evidence concerning the occurrence of coelenterazine and luciferase in other types of marine organisms establishes that coelenterazine is required for bioluminescence of various, distantly related, types of organisms such as squids, shrimps and fishes. 2. 2. In the bioluminescent shrimps and fishes, coelenterazine occurs most abundantly along the digestive tract, suggesting the possibility that this compound derives from ingested food.

Cypridina Bioluminescence: Light-Emitting Oxyluciferin-Luciferase Complex

Fluorescence of Cypridina oxyluciferin is greatly enhanced when it is bound to luciferase; the spectrum is thereby shifted, so that it corresponds precisely to the emission spectrum characteristic of the bioluminescentoxidation of luciferin. Thus the oxyluciferin-luciferase complex is the lightemitter. The binding is equimolar, with dissociation constant KD equal to 3x 10-7 mole per liter. The molecular weight of the luciferase, according to three different methods, is between 52,000 and 57,000; molecular activities of luciferase for the bioluminescence reaction and for the hydrolysis of oxy-luciferin are 1600 and 2 per minute, respectively.

MECHANISMS IN THE QUANTUM YIELD OF CYPRIDINA BIOLUMINESCENCE

Abstract— –The influence of temperature, pH, salts, and reactant concentrations on the biolumin‐escent oxidation of Cypridina luciferin catalyzed by Cypridina luciferase indicates a highest quantum yield φ (einsteins per mole of luciferin oxidized) of 0.31 in H2O, or 0.33 in 99% D2O. With the aid of data on fluorescence of the light‐emitting oxyluciferin‐luciferase complex, and of oxyluciferin in diglyme, partial explanations are suggested for the observed variations in φ, including the relatively low φ, of 0.03 for chemiluminescence of luciferin in organic solvents, wherein a different pathway of luciferin degradation, as indicated by chromatographic evidence, results in much less population of the excited state.

Further data on the specificity of aequorin luminescence to calcium

Abstract The possible triggering of luminescence of the photoprotein aequorin by 11 kinds of metal ions, in concentrations from 10 −3 to 10 −6 M, at pH 6.0 and at pH 8.0, were examined under conditions which minimized contamination with extraneous Ca 2+ . Y 3+ and La 3+ were found to have activities nearly as great as that of Ca 2+ at pH 6.0, but gave evidence of quenching effects in the higher concentrations at pH 8.0. Cations indicating no activity, or only negligible activity at either pH 6.0 and 8.0 included Be 2+ , Ba 2+ , Co 2+ , Ni 2+ , and Cu 2+ . Considerable activity was shown by Pb 2+ and Cd 2+ at pH 6.0, but very little at pH 8.0.

POLYNOIDIN: A MEMBRANE PHOTOPROTEIN ISOLATED FROM THE BIOLUMINESCENT SYSTEM OF SCALE‐WORMS

Abstract— In order to isolate and purify the luminescence system of scale‐worms, the scales were homogenized and extracted in the presence of Triton X‐100. After chromatography on Bio‐Gel A‐5m, Sephadex G‐75 and DEAE‐cellulose, a single peak in luminescence activity was obtained. It showed properties of a membrane protein having a high mol. wt (about 500000) with characteristics of a photoprotein. The photoprotein, for which we suggest the name polynoidin, emits light in response to several reagents that can produce superoxide or hydroxyl radicals, such as H2O2 plus Fe2+, but the luminescence is not triggered by Ca2 +. Oxygen is an absolute requirement for the luminescent reaction. The luminescence has a maximum at 510 nm. The photoprotein is not fluorescent when excited at 350 nm either before or after the luminescent reaction, thus differing distinctly from the green‐fluorescent riboflavin in photosomes which is easily separated at the first step of the purification. We suggest a mechanism of the in vivo luminescence of scale worms in which the production of superoxide or hydroxyl radicals by the oxidation of reduced riboflavin could be regulated by Ca2+.

Dinoflagellate luciferin is structurally related to chlorophyll

Among the several genera of dinoflagellates thus far examined, the luciferins and luciferases are crossreactive [3-51, thus indicating identity or close similarity in their luciferins. Recent studies have suggested that the cross-reactive luciferin of Pyrocystis hula, a non-motile, bladder-like, open ocean dinoflagellate, is an open chain polypyrrole, possibly similar to a bile pigment [6,7]. Chlorophyll (I was obtained from Sigma Chemical Co. (St Louis, MO) and pyrochlorophyll a produced by boiling chlorophyll a in pyridine for 24 h [lo]. Bilirubin was purchased from Serva Chemical (Heidelberg) and 1802 (99.1 atom %) and H2180 (90-91 atom %) from Prochem (Summit, NJ). ITLC Type SA silicic acid sheets for thin-layer chromatography (TLC) came from Gelman Instrument Co. (Ann Arbor MI). Ethyl ether and methanol were distilled before use.

Chaetopterus Photoprotein: Crystallization and Cofactor Requirements for Bioluminescence

The Chaetopterus photoprotein has been isolated in an amorphous form (molecular weight, 120,000) whlich in (NH4)2SO4 sollutionl converts to a crystalline form (molecular weight, 184,000) having the same specific light-emittinig activity; quantum yields are 0.0093 and 0.0143, respectively. Two new cofactor reqluirements have been separated fronm impure extracts: a macromnolecule resembling a nucleoprotein, and a lipid-like substance.

Chlorophyll-derived bile pigment in bioluminescent euphausiids

Most euphausiid shrimps are bioluminescent. The biochemical mechanism of luminescence, however, has been reported only for Meganyctiphanes nowegica [ 1,2]. In this euphausiid, light is emitted when a photoprotein is oxidized in the presence of a very unstable fluorescent substance, ‘F’. The compound F (M, < 1000, fluorescence emission maximum 476 mn) catalyzes the reaction and, at the same time, acts as the light-emitter of blue luminescence @,, 476 nm). These results strongly suggest that F, obtained from Meganyctiphanes norvegica and Euphausia pacifica, is a bile pigment-type compound derived from a chlorophyll. Although quantitatively more chlorophyll is degraded than heme in nature, virtually all bile pigments known are structurally degradation products of heme and very little has been known about bile pigments derived from chlorophylls. Compound F is a rare example of naturally occurring bile pigment that is chemically closely related to chlorophylls. Recently, the luciferin of luminous dinoflagellates was reported to be chemically similar to compound F [31.