Progress in the Study of Bioluminescent Earthworms

Even though bioluminescent oligochaetes rarely catch people's eyes due to their secretive lifestyle, glowing earthworms sighting reports have come from different areas on all continents except Antarctica. A major breakthrough in the research of earthworm bioluminescence occurred in the 1960s with the studies of the North American Diplocardia longa. Comparative studies conducted on 13 earthworm species belonging to six genera showed that N‐isovaleryl‐3‐aminopropanal (Diplocardia luciferin) is the common substrate for bioluminescence in all examined species, while luciferases appeared to be responsible for the color of bioluminescence. The second momentous change in the situation has occurred with the discovery in Siberia (Russia) of two unknown luminous enchytraeids. The two bioluminescent systems belong to different types, have different spectral characteristics and localization, and different temperature and pH optima. They are unique, and this fact is confirmed by the negative results of all possible cross‐reactions. The bioluminescent system of Henlea sp. comprises four essential components: luciferase, luciferin, oxygen and calcium ion. For Friderica heliota, the luminescent reaction requires five components: luciferase, luciferin, ATP, magnesium ion and oxygen. Along with luciferin, more than a dozen analogues were isolated from worm biomass. These novel peptide‐like natural compounds represent an unprecedented chemistry found in terrestrial organisms.     

Action spectra for photoinduced inactivation of bacteriophage T7 sensitized by 8-methoxypsoralen and angelicin

The action spectrum (240-300 nm) for photoinactivation of unsensitized phage T7 and the action spectra (310-380 nm) for photoinactivation of phage T7 sensitized with 8-methoxypsoralen (8-MOP) and angelicin were measured by an automated method. For unsensitized phage T7 the action spectrum is in good agreement with the absorption spectrum. For sensitization with angelicin the action spectrum is similar to the absorption spectrum, but for sensitization with 8-MOP the spectra are different. The agreement between the T7 absorption and action spectra in the far-UV region is due to photodamage of DNA, leading to phage inactivation. The similarity in the action and absorption spectra in the near-UV region for sensitization with angelicin seems to be in accordance with the monofunctional photobinding of angelicin to DNA. The action spectrum for sensitization with 8-MOP has a maximum at about 320 nm and this suggests that, in addition to the monoadducts, the biadducts play a role in the inactivation of phage T7. Taking the number of bound furocoumarin molecules into consideration, the quantum efficiencies were estimated. Furocoumarin increases the quantum efficiency in the near-UV region and the values are similar to those obtained in far-UV light without psoralens.     

A quantum chemical study of the formation of 2-hydroperoxy-coelenterazine in the Ca<Superscript>2+</Superscript>-regulated photoprotein obelin

The Ca²⁺-regulated photoprotein obelin determines the luminescence of the marine hydroid Obelia longissima. Bioluminescence is initiated by calcium and appears as a result of the oxidative decarboxylation related to the coelenterazine substrate. The luciferase of the luminescent marine coral Renilla muelleri (RM) also uses coelenterazine as a substrate. However, three proteins are involved in the in vivo bioluminescence of these animals: luciferase, green fluorescent protein, and Ca²⁺-regulated coelenterazine-binding protein (CBP). In fact, CBP that contains one strongly bound coelenterazine molecule is the RM luciferase substrate in the in vivo bioluminescent reaction. Coelenterazine becomes available for oxygen and the reaction with luciferase only after binding CBP with calcium ions. Unlike Ca²⁺-regulated photoproteins, the coelenterazine molecule is not activated by oxygen in the CBP molecule. In this work, by means of quantum chemical methods the behavior of substrates in these proteins is analyzed. It is shown that coelenterazine can form different tautomers: CLZ(2H) and CLZ(7H). The formation of 2-hydroperoxy-coelenterazine is studied. According to the obtained data, these proteins use different forms of the substrates for the reaction. In obelin, the substrate is in the CLZ(2H) form that affords hydrogen peroxide. In RM, coelenterazine is in the CLZ(7H) form, and therefore, CBP is not activated by oxygen.     

Photoinactivation Sensitivity of Staphylococcus carnosus to Visible-light Irradiation as a Function of Wavelength

Inactivation properties of visible light are of increasing interest due to multiple possible fields of application concerning antibacterial treatment. For violet wavelengths, the generation of reactive oxygen species by porphyrins is accepted as underlying mechanism. However, there is still little knowledge about photosensitizers at blue wavelengths. While flavins were named as possible candidates, there is still no experimental evidence. This study investigates the photoinactivation sensitivity of Staphylococcus carnosus to selected wavelengths between 390 and 500 nm in 10- to 25-nm intervals. Absorption and fluorescence measurements in bacterial lysates confirmed inactivation findings. By means of a mathematical calculation in MATLAB^®, a fit of different photosensitizer absorption spectra to the measured action spectrum was determined to gain knowledge about the extent to which specific photosensitizers are involved. The most effective wavelength for S. carnosus at 415 nm could be explained by the involvement of zinc protoporphyrin IX. Between 450 and 470 nm, inactivation results indicated a broad plateau, statistically distinguishable from 440 and 480 nm. This observation points to flavins as responsible photosensitizers, which furthermore seem to be involved at violet wavelengths. A spectral scan of sensitivities might generally be an advantageous approach for examining irradiation impact.     

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.     

KINETICS OF 4-THIOURIDYLATE-SENSITIZED PHOTOINACTIVATION OF RIBONUCLEASE A

Abstract —An action spectrum for the inactivation of ribonuclease A photosensitized with 4-thiouridylic acid follows the shape of the absorption spectrum of an RNase-thiouridylate complex or of the unbound nucleotide. The quantum yield is 3.3 times 10^-3. Statistical analysis of the data suggests that only the bound nucleotide participates in the photoinactivation. An Arrhenius plot for the first-order rate constants of inactivation gives a straight line in the range 0–40C and an apparent activation energy of photoinactivation of 5.7 kcal/mol. The 4-thiouridylate-sensitized photoinactivation of ribonuclease A is regarded as a kind of photodynamic action in which the substrate analogue plays the role of photosensitizer.     

Synthesis of Latia luciferin benzoate analogues and their bioluminescent activity

The bioluminescent system of the univalve shell Latia neritoides exhibits a luciferin-luciferase reaction. We study the enol formate structure of Latia luciferin, which is expected to be important for luminescent activity. The Latia luciferin analogues with an enol substituted benzoate moiety were synthesized and their bioluminescent activity was measured. The Latia luciferin benzoate analogues delay emission for natural luciferin in bioluminescence, indicating that the Latia bioluminescent activity can be controlled by the design of the enol ester.     

Tetracene oxygenation caused by infrared excitation of molecular oxygen in air-saturated solutions: the photoreaction action spectrum and spectroscopic parameters of the transition in oxygen molecules

The action spectrum of tetracene photooxygenation was measured in air-saturated carbon tetrachloride in the wavelength range of 1220–1290 nm using a wavelength-tunable forsterite laser. The data show that the photoreaction occurs due to laser excitation of the transition in oxygen molecules. The molar absorption coefficient (₁₂₇₃) and the cross section of light absorption (σ₁₂₇₃) corresponding to the spectral maximum of this transition were calculated from the observed photoreaction rates. The obtained values ε₁₂₇₃ = 0.003 M⁻¹ cm⁻¹ and σ₁₂₇₃ = 10⁻²³ cm² (±20%) reasonably correlate with those extrapolated from the high-pressure oxygen absorption spectra.     

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red‐shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red‐shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual‐color, far‐red to near‐infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far‐red to nIR emission maxima up to λ_max=706 nm with different Fluc mutants. This emission is the most red‐shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep‐tissue bioluminescence imaging.     

Novel Mechanism of Bioluminescence: Oxidative Decarboxylation of a Moiety Adjacent to the Light Emitter of Fridericia Luciferin

A novel luciferin from a bioluminescent Siberian earthworm Fridericia heliota was recently described. In this study, the Fridericia oxyluciferin was isolated and its structure elucidated. The results provide insight into a novel bioluminescence mechanism in nature. Oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as the light emitter.     

Theoretical Study of Firefly Luciferin pKa Values—Relative Absorption Intensity in Aqueous Solutions

Ground‐state vibrational analyses of firefly luciferin and its conjugate acids and bases are performed. The Gibbs free energies obtained from these analyses are used to estimate pK_a values for phenolic hydroxy and carboxy groups and the N–H⁺ bond in the N‐protonated thiazoline or benzothiazole ring of firefly luciferin. The theoretical pK_a values are corrected using the experimental values. The concentrations of these chemical species in solutions with different pH values are estimated from their corrected pK_a values, and the pH dependence of their relative absorption intensities is elucidated. With the results obtained we assign the experimental spectra unequivocally. Especially, the small peak near 400 nm at pH 1–2 in experimental absorption spectra is clarified to be due to the excitation of carboxylate anion with N‐protonated thiazoline ring of firefly luciferin. Our results show that the pK_a values of chemical species, which are contained in the aqueous solutions, are effective to assign experimental absorption spectra.     

Directed Improvement of Luciferin Regenerating Enzyme Binding Properties: Implication of Some Conserved Residues in Luciferin‐Binding Domain

Luciferin regenerating enzyme (LRE) contributes to in vitro recycling of d ‐luciferin to produce persistent and longer light emission by luciferase. Luciferin binding domains I and II among LREs regarded as potential candidates for luciferin‐binding sites. In this study, for the first time, amino acids T69, G75 and K77 located at luciferin binding domain I of LRE from L. turkestanicus (T‐LRE) substituted by using site‐directed mutagenesis. Single mutant T⁶⁹R increased luciferase light output more than two‐fold over a longer time in comparison with a wild‐type and other mutants of T‐LRE. Nevertheless, double mutant (K⁷⁷E/T⁶⁹R) increased the amount of bioluminescent signal more than two‐fold over a short time. In addition, G⁷⁵E, K⁷⁷E and G⁷⁵E/T⁶⁹R mutants did not improve luciferin–luciferase in vitro bioluminescence. Based on our results, addition of K⁷⁷E/G⁷⁵E and K⁷⁷E/G⁷⁵E/T⁶⁹R mutants caused intermediate changes in bioluminescence from in vitro luciferin–luciferase reaction. These findings indicated that the amino acids in question are possible to be located within T‐LRE active site. It may also be suggested that substituted Arg69 (Arg218) plays an important role in luciferin binding and the existence of Gly75 as well as Lys77 is essential for T‐LRE which has already evolved to have different functions in nature.     

Theoretical Study of Fluorescence Spectra Utilizing the pKa Values of Acids in Their Excited States

Assignment of the fluorescence spectrum of firefly luciferin in aqueous solutions was achieved by utilizing not only emission energies but also theoretical absorption spectra and relative concentrations as estimated by pK_a values. Calculated Gibbs free energies were utilized to estimate pK_a values. These pK_a values were then corrected by employing the experimental results. It was previously thought that the main peak near 550 nm observed in the experimental fluorescence spectra at all pH values corresponds to emission from the first excited state of the luciferin dianion [Ando et al. (2010) Jpn. J. Appl. Phys. 49, 117002–117008]. However, we found that the peak near 550 nm at low pH corresponds to emission from the first excited state of the phenolate monoanion of luciferin. Furthermore, we found that the causes of the red fluorescence at pH 1–2 are not only the emission from phenol monoanion but also the emission from the protonated species at nitrogen atom in the thiazoline ring of dianion.     

Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca2+‐regulated Photoproteins of Different Organisms

Upon binding their metal ion cofactors, Ca²⁺‐regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca²⁺‐regulated photoproteins—aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata—demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca²⁺‐regulated photoproteins.     

Larval Tenebrio molitor (Coleoptera: Tenebrionidae) Fat Body Extracts Catalyze Firefly D-Luciferin- and ATP-Dependent Chemiluminescence: A Luciferase-like Enzyme*

Ultraweak light emission was detected upon injection of firefly luciferin into live Tenebrio larvae. A chemilumi-nescent enzymatic activity dependent on molecular oxygen, D-luciferin and MgATP was then isolated from larval fat body extracts by precipitation with 70% ammonium sulfate. D-Luciferin and ATP can be replaced by luciferyl-adenylate. Pyrophosphate is a main product from the chemiluminescent reaction. The in vitro chemiluminescence intensity was not affected by peroxidase inhibitors such as N₃^?_- (0.5 mM) and CN^? (1 mM), attesting to its nonperoxidatic nature but was strongly inhibited by AMP (1 mM), luciferin 6′-ethyl ether (1 mM) and sodium pyrophosphate (2 mM), well-known firefly lucifer-ase inhibitors. Some physical-chemical properties of this enzymatic activity were similar to those of firefly lucif-erase (K_MATP = 195 μM; K_0.5 luciferin - 0.8 mM; optimum pH 8.5; δ_max= 610 nm at pH 8.5; firefly lucifer-ase: δ_max= 565 nm at pH 8.0 and 619 mm at pH 6.0), but the chemiluminescence was not affected by addition of polyclonal antibodies raised against Photinus pyralis luciferase. These data suggest that this chemiluminescence results from a ligase with luciferase activity.     

TDDFT Study of the Electronic Structure,Absorption and Emission Spectra of the Light Emitters of the Amazing Firefly Bioluminescence and Solvation Effects on the Spectra

The ground and excited state properties of luciferin (LH₂) and oxyluciferin (OxyLH₂), the bioluminescent chemicals in the firefly, have been characterized using density functional theory (DFT) and time dependent DFT (TDDFT) methods. The effects of solvation on the electronic absorption and emission spectra of luciferin and oxyluciferin were predicted with a self‐consistent isodensity polarized continuum model of the solvent using TDDFT. The S₀→S₁ vertical excitation energies in the gas phase and in water were obtained. Optimizations of the excited state geometries permitted the first predictions of the fluorescence spectra for these biologically important molecules. Shifts in both of the absorption and emission spectra on proceeding from the gas phase to aqueous solution were also predicted.     

LC-MS and microscale NMR analysis of luciferin-related compounds from the bioluminescent earthworm Fridericia heliota

This paper presents the main results of RP-HPLC-MS and microscale NMR analysis performed on Accompanying similar to Luciferin (AsLn(x)), compounds present in extracts of the bioluminescent earthworm Fridericia heliota that display similarities with Fridericia's luciferin, the substrate of the bioluminescent reaction. Three isomers of AsLn were discovered, AsLn(1), AsLn(2) and AsLn(3), all of which present a molecular weight of 529 Da. Their UV-Vis absorption spectra show maxima at 235 nm for AsLn(1), 238 and 295 nm for AsLn(2) and 241 and 295nm for AsLn(3). MS(n) fragmentation patterns suggest the existence of carboxylic acid and hydroxyl moieties, and possibly chemical groups found in other luciferins like pterin or benzothiazole. The major isomer, AsLn(2), presents an aromatic ring and alkene and alkyl moieties. These luciferin-like compounds can be used as models that could give further insights into the structure of this newly discovered luciferin.     

Purification and partial spectral characterization of a novel luciferin from the luminous enchytraeid Fridericia heliota

A homogeneous luciferin preparation has been obtained from the luminous soil enchytraeid Fridericia heliota, which has an ATP-dependent luminescent system. A procedure for luciferin purification without losing fractions of active luciferase has been developed. The luciferin specific activity is 4000 times increased; its UV absorption spectrum maximum is 294 nm with a local minimum at 262 nm. The luciferin of the enchytraeid F. heliota is significantly different from firefly luciferin, whose luminescent reaction also requires ATP, and it also appears to have no similarities to other known luciferins.     

Spectral characteristics of the photoreceptor pigment of phototaxis in Dictyostelium discoideum

Abstract —A photoresponsive pigment, described previously as the photoreceptor pigment for phototaxis in Dictyostelium discoideum, was examined by low-temperature spectroscopy. The absorption spectrum of the purified pigment frozen in darkness indicates the reduced form of a high-spin heme protein, and the spectrum of the pigment frozen during irradiation (to freeze in the photoproduct) indicates the oxidized form of the heme protein. The light-induced absorbance changes measured at room temperature also indicate photooxidation of a heme pigment. The action spectrum for the light-induced absorbance change shows a primary peak at about 430 nm and a broad, less active maximum in the 550–580 nm region. The absorption spectrum of the reduced pigment and the action spectrum for its photooxidation are both similar to the action spectrum for phototaxis of the pseudoplasmodia of D. discoideum.