Analysis of Organotin Compounds via a Thermabeam® LC–MS Interface

Selected organotin compounds, relating to antifouling paints, have been analysed using a particle beam interface system designed for use on liquid chromatography–mass spectrometry (LC–MS) instruments. The resultant mass spectra matched those obtained from conventional electron-impact (EI) techniques, and consistent data over several injections and different elution times were obtained. Data obtained from tributyltin, dibutyltin, monobutyltin, triphenyltin and diphenyltin (each as the chlorides) are presented. This interface has been shown to maintain sample and therefore spectral integrity for these compounds and is of potential use in further investigations relating to organotin environmental pollution.     

EVIDENCE FOR HIGH ENERGY STORAGE INTERMEDIATES IN BIOLUMINESCENCE

Abstract— The bioluminescent enzyme from Photobacterium fischeri is normally activated in vifro by reaction with FMNH₂ and O₂. in the presence of a long-chain aldehyde. Emission from enzyme intermediates in this reaction continues for several seconds, and if the mixture is frozen just after initiation of the reaction, this presumptive emission may be delayed until the system is warmed again. Light is then emitted in a fashion analogous to thermo-luminescent emission, with a maximum intensity at — 10°C. The experiments described here show that the total amount of light which is emitted under these conditions no longer depends so much upon aldehyde, a relatively high quantum yield being obtained both with and without aldehyde.
It is further shown that bioluminescence may be activated by light, populating it is believed, the same state which is responsible for the emission in the case of the FMNH₂-induced emission. The light-induced reaction does not depend on flavin in the enzyme preparations, nor does the activation spectrum resemble that of a flavoprotein. Activation may be carried out in the solid state at temperatures down to at least — 100°C, and so does not involve the diffusion of large molecules. It is proposed that energy storage takes place by charge separation, and that the excited state from which emission takes place is associated with charge recombination.     

Das ?therische Zimmt?l

Ohne Zusammenfassung     

A BLUE FLUORESCENT PROTEIN FROM A YELLOW-EMITTING LUMINOUS BACTERIUM

Vibrio fischeri strain Y1 emits yellow light in vivo due to the participation of a yellow fluorescent protein (YFP) in the luciferase reaction. In this study it was found that the organism also produces a protein (referred to as Y1-BFP) emitting strong blue fluorescence. Its molecular weight, about 25 kDa, is the same as or very close to that of YFP. The fluorescence excitation and emission maxima of the purified Y1-BFP are at 416 and 461 nm, respectively, and the fluorescence lifetime is 12.5 ns at 2 degrees C. The molar extinction coefficient of Y1-BFP at 416 nm was estimated to be approx. 9500. With the homologous luciferase, Y1-BFP decreases the intensity and rate of decay in the in vitro reaction but has no effect on its emission spectrum (in contrast to YFP, which has a striking effect on the spectrum). With luciferase isolated from Vibrio harveyi, however, Y1-BFP causes a small blue-shift (approximately 10 nm) in the emission of the enzyme catalyzed reaction, whereas YFP has no effect on the emission spectrum.     

Effectiveness of the Accessory Yellow Fluorescent Protein in the Bacterial Luciferase Reaction Correlates with the Lifetime of the Peroxyhemiacetal Intermediate: The Stereochemistry of the Reaction

In the in vitro reaction of Vibrio fischeri Y-1 luciferase, the dependence of the initial luminescence intensity (I_o) and its rate of decay (k_d) on the chain-length of the aliphatic aldehyde are greatly altered by the presence of yellow fluorescent protein (YFP), which functions as an accessory emitter. Whereas with no YFP both k_d and I_o are maximum for chain lengths ≥ 12, the fraction of the light emitted from the accessory chromophore, measured as the ratio of yellow to blue light (Y/B), is greater with shorter chain-length aldehydes. Thus, aldehydes that are least efficient in the absence of YFP are more efficient for causing yellow emission in its presence. These results are interpreted on the basis of the expected lifetimes of the peroxyhem-iacetals with which YFP interacts: high values of k_d reflect short peroxyhemiacetal lifetimes, hence less chance of interaction with YFP. The critical dependence on aldehyde chain-length underlines the importance of stereochemical factors in the bacterial reaction, which are discussed here in the framework of a chemically initiated electron exchange luminescence model.     

BORONIC ACIDS AS MECHANISTIC PROBES FOR THE BACTERIAL LUCIFERASE REACTION

Bacterial luciferase uses long chain aldehydes as substrates. Alkylboronic acid analogs of these substrates with chain lengths of C₇ and C₉ have been synthesized, characterized, and used as mechanistic probes for the light emitting reaction. They behave as inhibitors in the in vitro luminescence reaction. Contrary to an earlier report (Macheroux and Ghisla, 1985, Nachr. Chem. Tech. Lab. 33,785–790) they are not substrates for bacterial luciferase, in that they do not lead to light emission and are not oxidized by the flavin-4a-hydroperoxide to the products boric acid and the corresponding alcohol, as would be expected from a Baeyer-Villiger reaction. However, the particular conformation of a putative boronic acid hydroperoxide at the active center might be such that it would preclude a Baeyer-Villiger fragmentation. Thus, while the results do not support the postulate that luciferase proceeds via a Baeyer-Villiger mechanism, they also do not exclude it. A further observation was that the endogenous light emission (no added aldehyde) decays more rapidly than does the luciferase bound flavin-4a-hydroperoxide. This suggests that the endogenous light is not caused by the decomposition of the flavin-4a-hydroperoxide.     

Two bioluminescent diptera: the North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (lambda(max) = 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin-luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of approximately 140 kDa and an additional high molecular weight fraction (possibly a luciferin-binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin-luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.     

High-pressure phase transition in CaTiOSiO4 titanite

The P2₁/a to A2/a transition in titanite has been studied at high pressure (and room temperature) by X-ray powder diffraction. On the basis of the disappearance of k + l= odd reflections from the diffraction pattern, the transition was located between 3·351(3) and 3·587(3) GPa. The variation with pressure of the spontaneous strain in the P2₁/a phase indicates that the transition has an effective critical exponent significantly less than 1/2. Within the uncertainties of the data, the transition could be weakly first-order in character or be continuous with an effective critical exponent in the range of ～0·13-0·25. The A2/a to P2₁/a transition is accompanied by a significant expansion of the a-axis as a result of the bond-valence sum requirements of the Ti atoms: in the high-pressure phase they occupy the centres of the TiO₆ octahedra, but they are displaced along the a-axis in the P2₁/a phase to form alternating short and long Ti-O bonds.     

Electrospray Mass Spectrometry: An Alternative Method for the Identification of Organotin Compounds

The limitations of conventional gas chromatography–mass spectrometry (GC–MS) analyses for alkyl- and aryl-tin compounds are discussed, particularly the excessive fragmentation from electron impact (EI) ionization. Negative EI methods exhibit low ionization capabilities and are restricted to compounds with an electronegative centre, and are thus not suitable for general routine analysis. Liquid chromatography–MS (LC–MS) interfaces offer potential advantages in terms of reduced sample work-up since no derivatization is required. Electrospray techniques give reproducible mass spectra for each compound studied under fixed instrumental parameters. Changes in the cone/repeller voltages can radically alter the observed mass spectra. High-mass species were observed for each compound studied and tentative structures for these species are proposed.