BIOCHEMISTRY OF CENTIPEDE BIOLUMINESCENCE*

Abstract— The centipede (Orphaneous brevilabiatus) secretes a bioluminescent slime. The corrected emission spectrum of this luminescence was found to have maxima at about 510 and 480 nm. The reaction was found to require both a luciferin and luciferase and showed an unusually low pH optimum (4.6). Oxygen was required for the reaction, but oxygen could interact with one of the components allowing for anaerobic light emission.     

BIOLUMINESCENCE OF ACETALDEHYDE CATALYZED BY BACTERIAL LUCIFERASE

Abstract— Bioluminescence from the oxidation of acetaldehyde by bacterial luciferase of Photobacterium fischeri has been demonstrated for the first time and its efficiency has been compared with that of nonanal and other short chain aldehydes.     

KINETICS OF BACTERIAL BIOLUMINESCENCE AND THE FLUORESCENT TRANSIENT

Abstract— The addition of FMNH₂ to Vibrio harveyi luciferase at 2°C in the presence of tetradecanal results in the formation of a highly fluorescent transient species with a spectral distribution indistinguishable from that of the bioluminescence. The bioluminescence reaches maximum intensity in 1.5 s and decays in a complex manner with exponential components of 10^-1s^-1, 7 × 10^-3s^-1, and 7 × 10 ⁴s^-1. The fluorescent transient rises exponentially at 7 × 10^-2s^-3 and decays at 3 × 10^-4s^-1. The slowest bioluminescence component, comprising the bulk of the bioluminescence, decays at twice the rate of the fluorescent transient under all variations of reaction conditions: concentration of reactants, temperature 2–20°C, and aldehyde chain length—decanal, dodecanal and tetradecanal. The activation energy for both the slowest bioluminescence decay and the transient fluorescence decay is 80 kJ-mol^-1. An energy transfer scheme is proposed to explain the results where two distinct chemically energized species utilize the fluorescent transient as emitter for the slower bioluminescences, and for the faster process a fluorophore present in the protein preparation. Kinetic observations suggest that typical preparations of V. harveyi luciferase comprise 15% active protein.     

SENSITIZATION BY LUMAZINE PROTEINS OF THE BIOLUMINESCENCE EMISSION FROM THE REACTION OF BACTERIAL LUCIFERASES

Abstract— Lumazine protein from Photobacterium phosphoreurn blue shifts the in vitro bioluminescence spectra in the reactions using each of the 4 main types of bacterial luciferases: P. phosphoreum, P. leiognathi, Vibrio harveyi and V. fischeri . For the reaction initiated with FMNH₂ and tetradecanal at 2°C, this "sensitizing" property of lumazine protein differs quantitatively between the luciferases. An interaction constant characterizing each type of luciferase may be derived from a reciprocal plot of the spectral shift against the lurnazine protein concentration. The weakest interaction constant is in the V. fischeri reaction, 180 μM. For the V. harveyi reaction the interaction is in the range 6–9 μ M , and for both Photobacterium reactions it is 2–3 μM. A concentration of only 0.6 μ M of lumazine protein is sufficient to cause an observable change in the Photobacterium bioluminescence spectra. For the V. harveyi case the interaction constant is near to the equilibrium K _d for the luciferase-lumazine protein complex, observed directly by Visser and Lee. Both constants are decreased markedly by increase in phosphate concentration so that it is concluded that, with V. harveyi luciferase, sensitization occurs within this protein-protein complex. For P. phosphoreum luciferase, however, the equilibrium complex is too weak to correspond to the sensitizing interaction and it is concluded that the rate-limiting process is a protein-protein bimolecular collision. As judged from their molecular weight around 20000, spectral properties, and ability to blue shift the bioluminescence spectra, lumazine proteins are identified in a second strain of P. phosphoreum and in P. leiognathi .     

DITHIONITE TREATMENT OF FLAVINS: SPECTRAL EVIDENCE FOR COVALENT ADDUCT FORMATION and EFFECT ON in vitro BACTERIAL BIOLUMINESCENCE

Intrigued by the apparent requirement of dithionite for FMN reduction (as opposed to photoreduction or catalytic hydrogenation) in the H2O2-initiated bacterial bioluminescence reaction, we chose 5-ethyl-3-methyllumiflavinium cation I as a model to investigate possible flavin adduct formation by treatment with dithionite or (bi)sulfite. In the range of pH 5-8, the reaction of dithionite with 5-ethyl-3-methyllumiflavinium cation, which is in equilibrium with the 5-ethyl-4a-hydroxy-3-methyl-4a, 5-dihydrolumiflavin pseudobase II (X = OH), is not limited to the formation of flavosemiquinone and dihydroflavin following two one-electron steps. Several parallel and sequential reactions may take place involving the intermediacy of covalent flavin adducts. Addition of (bi)sulfite gave a 4a-sulfiteflavin adduct II (X = SO3-). Consistent with the S2O4(2-) in equilibrium with 2 SO2-. equilibrium, the reaction of dithionite and II (X = OH; SO3-) gave rise to two flavin adducts in competitive nucleophilic displacements: a 4a-sulfoxylate-flavin radical (II, X = SO2.) and a 4a-dithioniteflavin adduct (II, X = S2O4-), respectively. On increasing the (S2O4(2-), SO2.-)/flavin ratio under N2, the formation of the 4a-sulfoxylate-flavin radical became predominant. The II (X = SO2.) so formed was in equilibrium with the flavosemiquinone and bisulfate and can be trapped by reacting with hydroxylamine. In the initial presence of oxygen, II (X = SO2.) was highly reactive toward O2, giving a fast oxidation to II (X = SO3-) and effectively suppressing the formation of the flavosemiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE ORIGIN OF BIOLUMINESCENCE*

Abstract— Primitive luciferases evolved in order to utilize oxygen directly as an electron acceptor at the low oxygen concentrations of the primitive atmosphere. This provided a major selective advantage in the ability to metabolize aromatic molecules and n-alkanes. These strongly exergonic reactions produce product molecules in electronically excited states, from which light emission is possible. The low-level luminescence observed from microsomal extracts, from the action of leukocytes on phagocytized bacteria and from rapidly growing tissues is ascribed to these same exergonic hydroxylase reactions, or to the release during these reactions of superoxide radicals which can initiate chemiluminescent reactions. Bioluminescence is a later secondary adaptation of this oxygenase reaction to signaling for sex, food or escape. The similarity of the reaction pathways is shown in the three bioluminescent systems from which the substrates and products have been identified.     

THE EVOLUTION OF BIOLUMINESCENCE IN BACTERIA

Individuals of aerobic, saprophytic bacterial species, encountering a changing environment in which they were regularly exposed to periods of severe hypoxia, would have gained a major selective advantage in fatty acid metabolism if a mutation in a flavoprotein oxygenase permitted them to oxidize accumulated C₈-C₁₈ aliphatic aldehydes to fatty acids at oxygen concentrations below which the cytochrome oxidase mediated electron transport pathway became inhibited. Such mutants, able to continue to metabolize exogenous lipid fatty acids under hypoxia, might have outproduced their wild type ancestors. Colonies of those mutants which utilized reduced flavin mononucleotide (FMNH₂) as the flavin cofactor would have been luminous, owing to the fortuitous coincidence that the aldehyde oxygenation resulted in an enzyme-flavin excited electronic state which emitted blue light with a high fluorescence yield. If this accidental “proto-bioluminescence” were initially of sufficient brightness to have elicited phototactic responses in nearby motile organisms, increasing thereby detrital food sources or the potential for dispersal and colonization for the bacteria, a new and completely different selective advantage, that of bioluminescent signalling, would have arisen from the original metabolic function. This is the biochemical analog of Darwin's principle of functional change in structural continuity. It is proposed that bacterial luciferase, the FMNH₂-oxygenase in luminous bacteria, was such a mutation. The present ubiquitous distributions of luminous bacterial species in marine waters, on the surfaces of marine animals and as symbionts in the specialized light organs and digestive tracts of many fish species have resulted from subsequent environmental selection and optimization for this original proto-bioluminescent reaction. By extension it is suggested whereas “protobioluminescence” arose in many species independently whenever metabolic oxygenation of a substrate resulted in an adventitiously efficient chemiluminescence, the function of bioluminescence arose only upon favorable interaction between the “proto-bioluminescence” and its ecosystem.     

APPLICATION OF PHOTOSENSITIVE DEVICES TO BIOLUMINESCENCE STUDIES

Abstract—A brief review is given of some results obtained by the application of image intensification to studies of bioluminescence. The system consists of an image intensifier placed at the output of a suitable microscope., so that the image from the microscope falls on the intensifier cathode. The photon gain of the intensifier can be varied from a few thousand to one million. The output of the intensifier is recorded either on film or, in most applications to date, by means of a TV vidicon. The TV system permits display on a monitor in real time and simultaneous recording on magnetic tape for subsequent playback and analysis. It also provides time resolution for dynamic studies. Results are summarized for in vivo observations on Noctiluca miliaris, Obelia, Renilla , and Mnemiopsis leidyi . Utilization of the luminescence of aequorin in the presence of Ca²⁺ has been directed to observations on amoebae and the egg of the Medaka fish.
Studies at the molecular level have been made by means of the spectral distribution of the output light. In these, the output of a fast input lens grating spectrometer is focused on the image intensifier cathode. Thus the entire visible spectrum of an in vivo bioluminescent flash can be intensified and recorded on film by photographing the output. The film is then analyzed by means of a digitized densitometer, and a computer program corrects the observed spectrum for system non-linearities and non-uniformities. In this way, the in vivo spectra of 15 bioluminescent species have been recorded.     

ON THE MECHANISM OF PHAUSIS SPLENDIDULA BIOLUMINESCENCE

Evidence is presented for cross reactivity between the luciferases and luciferins of Phausis splendidula and Photinus pyralis.     

BIOLUMINESCENCE OF THE BRITTLE STAR OPHIOPSILA CALIFORNICA

The light-emitting principle of the brittle star Ophiopsila californica has been isolated and purified. It was found to be a green-fluorescent photoprotein (molecular weight 45000) which emits green light (λ_max 500 nm) when H₂O₂ is added, independently of the presence or absence of O₂. The green fluorescence (emission maximum 500 nm, excitation maximum 440 nm) spectrally coincided with the H₂O₂-triggered luminescence, indicating that the green fluorescent chromophore is the light-emitter of the photoprotein luminescence.     

PHOTOINHIBITION OF STIMULABLE BIOLUMINESCENCE IN MARINE DINOFLAGELLATES

Abstract— Different genera of bioluminescent photosynthetic dinoflagellates exhibit different mechanisms for the inhibition of stimulable bioluminescence during daylight. These are (a) reduction in bioluminescence capacity, (b) increased refractoriness to mechanical stimulation, and (c) inhibition of transmission of signals from mechanical receptor sites to bioluminescence emission sites. The increase in stimulable bioluminescence that in nature mirrors the decrease in sunlight intensity prior to sunset is dependent upon the logarithm of the ambient irradiation intensity. Photoinhibition of bioluminescence in all species examined except Gonyaulax polyedra is the result of absorption of light in the blue region of the spectrum.