Spectroscopic properties of protochlorophyll forms in Triton X-100 detergent micelles

Protochlorophyll (PChl) forms were performed in Triton X-100 detergent micelles. The concentration of Triton X-100 was 7·10^?4 M (above the critical micellar concentration); the concentration of PChl varied between 1.6·10^?5 and 1.8·10^?4 M. Absorption, fluorescence and circular dichroism (CD) spectra were registered. The absorption spectra were resolved into Gaussian components by computer analysis. PChl forms with absorption bands at 632–634, 638, 652–654, 663–664, 668 and 676 nm and with fluorescence emission bands at 634–636, 640–644, 652–655, 677–678, 686 and 694–696 nm were observed in micellar solutions of different PChl concentrations. The CD spectra showed a strong dependence on the concentration of PChl: positive CD signals or positive Cotton effects were observed in the vicinity of 650 nm. The intensity of these signals increased in parallel with increasing concentration of PChl. No CD signals were found in the region of the longer wavelength absorption bands. These data show that the PChl exists in many different forms in this system, and the spectroscopic properties of these forms are determined by different molecular interactions viz., interactions of PChl with Triton X-100 or water molecules and/or by the aggregation of PChl.     

Electric field promotion of the bacteriorhodopsin BR570 to BR412 photoconversion in films of Halobacterium halobium purple membranes

The combined action of electric field (10⁵–10⁷ V · m^?1) and light (380–580 nm, 80 W · m^?2) activating the photoenergetic reaction of bacteriorhodopsin (BR) in dry films of purple membranes from Halobacterium halobium was studied. A new stimulating effect of the field on the BR₄₁₂ intermediate accumulation in the normal photochromic cycle of BR₅₇₀ has been observed. The formation of the product BR₄₁₂ is supposed to be accompanied by specific rearrangements of certain charged, polar and polarizable groups in the BR pigment-protein matrix. Such an intrinsic polarization could be promoted by an external electric field, the displacement vector of those groups being oriented in the direction of the field. The dielectric polarization properties of the purple membranes have been demonstrated by electret-thermal analysis.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10¹³ photons · cm^?2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10¹³–10¹⁶ photons · cm^?2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Fluorimetry of mitochondria in cells vitally stained with DASPMI or rhodamine 6 GO

The fluorescent dyes DASPMI and rhodamine 6 GO specifically stain mitochondria in living cells. Dye concentrations from 10^?8 to 5 × 10^?6 mole l^?1 can be used. Excitation and emission spectra, and quantum efficiency of DASPMI depend on solvent characteristics. Spectra of mitochondria in living cells correspond to those in phospholipids (excitation around 470 nm, emission 560–570 nm). Fluorescence intensity of DASPMI is a measure for the energization of mitochondria, as revealed by in vitro studies. In living cells uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation (i.e. by oligomycin, FCCP). Distribution of fluorescence intensity indicates an intracellular gradient in energy load of endothelial cells. Single mitochondria exhibit oscillations in fluorescence. Mitochondria loaded with DASPMI release the dye suddenly into the cytoplasm on treatment with FCCP. Cyanide and antimycin however, do not diminish fluorescence in vivo under optimal nutritional conditions, while they do so in mitochondrial suspension, indicating different mitochondrial behaviour in vivo and in suspension.     

Synthesis and properties of a fluorescent derivative of phosphatidylcholine

1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidyl choline, (NBD-PC) was prepared by alkylation of ?-amino caproic acid with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1), followed by esterification of lysophosphatidylcholine. The compound was purified by silicic acid chromatography, and exhibited a single spot on thin layer chromatography using acidic, basic and neutral solvent systems, when visualized by UV, molybdate spray, primuline, or charring. The UV-visible absorption spectrum, and the uncorrected fluorescence excitation spectrum of NBD-PC in absolute ethanol showed maxima at approximately 340 and 460 nm, while the fluorescence emission spectrum showed a single peak at 525 nm. Fluorescence intensity and emission maximum wavelength of NBD-PC are strongly dependent on solvent dielectric constant, and the relative fluorescent intensity of NBD-PC in absolute ethanol is directly proportional to its concentration from 1 ng/ml to approximately 3 μg/ml. Incorporation of NBD-PC into membranes of human lymphocytes cultures in the presence or absence of phytohemagglutinin (PHA) resulted in a marked increase in the relative fluorescent intensity of the bound fluorochrome, and a 15 nm blue shift in its emission maximum wavelength. Fluorescence titration data indicate that the unstimulated lymphocytes bound 912 pmoles NBD-PC/mg protein with an association constant of 3.45 × 10⁷ M, while the PHA stimulated cells bound 1200 pmoles NBD-PC/mg protein with an association constant of 2.82 × 10⁷ M. The temperature dependence of the fluorescent intensity of NBD-PC incorporated in control, and PHA stimulated lymphocytes showed discontinuities at 15 and 24 °C respectively. Fluorescence polarization of NBD-PC incorporated in the membranes of stimulated lymphocytes was greater than the polarization of the fluorochrome in non-stimulated cells, suggesting that the plasma membranes of PHA stimulated lymphocytes contain regions of higher microviscosity.     

Contribution of the Resonance Raman Spectroscopy to the Identification of Z DNA

Abstract

Poly(dG-dC)?poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition.

With 257 nm excitation wavelength the 1580 cm^?1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm^?1 and at 1193 cm^?1 and the bands of the cytosines at 780 cm^?1, 1242 cm^?1 and 1268 cm^?1.

By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.     

Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans-bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum (λ_max) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ_max was located at 610 nm and the extinction coefficient (?_max) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ_max at 596 nm and an ?_max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ_max at 543 nm and an ?_max 0.66-times that of bacteriorhodopsin.     

Eu2+‐induced enhancement of defect luminescence of ZnS

The Eu²⁺‐induced enhancement of defect luminescence of ZnS was studied in this work. While photoluminescence (PL) spectra exhibited 460 nm and 520 nm emissions in both ZnS and ZnS:Eu nanophosphors, different excitation characteristics were shown in their photoluminescence excitation (PLE) spectra. In ZnS nanophosphors, there was no excitation signal in the PLE spectra at the excitation wavelength λ_ex > 337 nm (the bandgap energy 3.68 eV of ZnS); while in ZnS:Eu nanophosphors, two excitation bands appeared that were centered at 365 nm and 410 nm. Compared with ZnS nanophosphors, the 520 nm emission in the PL spectra was relatively enhanced in ZnS:Eu nanophosphors and, furthermore, in ZnS:Eu nanophosphors the 460 nm and 520 nm emissions increased more than 10 times in intensity. The reasons for these differences were analyzed. It is believed that the absorption of Eu²⁺ intra‐ion transition and subsequent energy transfer to sulfur vacancy, led to the relative enhancement of the 520 nm emission in ZnS:Eu nanophosphors. In addition, more importantly, Eu²⁺ acceptor‐bound excitons are formed in ZnS:Eu nanophosphors and their excited levels serve as the intermediate state of electronic relaxation, which decreases non‐radiative electronic relaxation and thus increases the intensity of the 460 nm and 520 nm emission dramatically. In summary, the results in this work indicate a new mechanism for the enhancement of defect luminescence of ZnS in Eu²⁺‐doped ZnS nanophosphors. Copyright © 2016 John Wiley & Sons, Ltd.     

A study of the spectral and luminescence manifestations of the aggregation of thymine chromophores in polythymidylic acid aqueous solutions at room temperature in the context of photochemical information recording using thymine

The luminescence and excitation spectra of polythymidylic acid aqueous solutions at room temperature were studied. In addition to the previously described band at 338 nm, two new bands at 320 and 350 nm were recorded at various excitation wavelengths. An examination of the excitation spectra that had not been studied previously, as well as their comparison with the differential absorption spectra previously recorded during photodimerization, allowed us to interpret the band at 320 nm as the band of noninteracting chromophores; the band at 338 nm as the band of the most photochemically active, densely packed stacking dimmers (exciton splitting of absorption band of ～4000 cm^?1); and the band at 350 nm as the band of photochemically inactive large stacking aggregates (n ≥ 10, exciton splitting of ～8000 cm^?1). The changes in the optical density of the polythymidylic acid aqueous solution at γ = 270 nm after successive irradiation of the solution with light at 279 + 302 and 248 nm were studied. The reasons for their incomplete reversibility are discussed.     

Development of an optical sensor for chlortetracycline detection based on the fluorescence quenching of l‐tryptophan

A simple and selective spectrofluorimetric method for the detection of chlortetracycline (CTC) was studied. In pH 7.4 buffer medium l ‐tryptophan (l ‐Trp), applied as the fluorescence probe, interacted with CTC resulting in fluorescence quenching of the probe. CTC was detected with maximum excitation and emission wavelengths at λ_ex/λ_em = 275/350 nm. Notably, quenching of fluorescence intensities was positively proportional to the CTC concentration over the range of 0.65–30 μmol L^?1 and the limit of detection was 0.2 μmol L^?1. Effect of temperature shown in Stern?Volmer plots, absorption spectra and fluorescence lifetime determination, indicated that fluorescence quenching of l ‐Trp by CTC was mainly by static quenching. The proposed study used practical samples analysis satisfactorily.     

BIO-OPTICAL CHARACTERISTICS AND PHOTOADAPTIVE RESPONSES IN THE TOXIC AND BLOOM-FORMING DINOFLAGELLATES GYRODINIUM AUREOLUM,GYMNODINIUM GALATHEANUM,AND TWO STRAINS OF PROROCENTRUM MINIMUM1

Photoadaptive responses in the toxic and bloom-forming dinoflagellates Gyrodinium aureolum Hulbert, Gymnodinium galatheanum Braarud, and two strains of Prorocentrum minimum (Pavillard)Schiller were evaluated with respect to pigment composition, light-harvesting characteristics, carbon and nitrogen contents, and growth rates in shade- and light-adapted cells. The two former species were grown at scalar irradiances of 30 and 170 μmol · m ^?2 at a 12-h daylength at 20° C. The two strains of P. minimum were grown at 35 and 500 μmol. m^?2· s^?1 at a 2-h daylength at 20° C. For the first time, chlorophyll (chl) c₃, characteristic of several bloom-forming prymnesiophytes, was detected in G. aureolum and G. galatheanum. Photoadaptional status affected the pigment composition strongly, and the interpretation of the variation depended on whether the pigment composition was normalized per cell, carbon, or chl a. Species-specific and photoadaptional differences in chl a-specific absorption (°a_c, 400–700 nm) and chl a-normalized fluorescence excitation spectra of photosystem II fluorescence with or without addition of DCMU (°F and °F_DCMU 400–700 nm) were evident. Gyrodinium aureolum and G. galatheanum exhibited in vivo spectral characteristics similar to chl c₃-containing prymnesiophytes in accordance with their similar pigmentation. Prorocentrum minimum had in vivo absorption and fluorescence characteristics typical for peridinin-containing dinoflagellates. Species-specific differences in in vivo absorption were also observed as a function of package effect vs. growth irradiance. This effect could be explained by differences in intracellular pigment content, cell size/shape, and chloroplast morphology/numbers. Light- and shade-adapted cells of P. minimum contained 43 and 17% of photoprotective carotenoids (diadino + diatoxanthin) relative to chl a, respectively. The photoprotective function of these carotenoids was clearly observed as a reduction in °F and °F ^DCMU at 400–540 nm compared to °ac in light-adapted cells of P. minimum. Spectrally weighted light absorption (normalized to chl a and carbon, 400–700 nm) varied with species and growth conditions. The use of quantum-corrected and normalized fluorescence excitation spectra with or without DCMU-treated cells to estimate photosynthetically usable light is discussed. The usefulness of in vitro absorption and fluorescence excitation spectra for estimation of the degradation status of chl a and the ratio of chl a to total pigments is also discussed.     

Sub-microsecond chlorophyll A delayed fluorescence from Photosystem I Magnetic field-induced increase of the emission yield

(1) In photosystem I (PS I) particles in the presence of dithionite and intense background illumination at 290 K, an external magnetic field (0–0.22 T) induced an increase, ΔF, of the low chlorophyll a emission yield, F ($\text{ΔF}\text{F}\text{? 1–1.5\%}$). Half the effect was obtained at about 35–60 mT and saturation occurred for magnetic fields higher than about 0.15 T. In the absence of dithionite, no field-induced increase was observed. Cooling to 77 K decreased ΔF at 685 nm, but not at 735 nm, to zero. Measuring the emission spectra of F and ΔF, using continuous excitation light, at 82, 167 and 278 K indicated that the spectra of F and ΔF have about the same maximum at about 730, 725 and 700 nm, respectively. However, the spectra of ΔF show more long-wavelength emission than the corresponding spectra of F. (2) Only in the presence of dithionite and with (or after) background illumination, was a luminescence (delayed fluorescence) component observed at 735 nm, after a 15 ns laser flash (530 nm), that decayed in about 0.1 μs at room temperature and in approx. 0.2 μs at 77 K. A magnetic field of 0.22 T caused an appreciable increase in luminescence intensity after 250 ns, probably mainly caused by an increase in decay time. The emission spectra of the magnetic field-induced increase of luminescence, ΔL, at 82, 167 and 278 K coincided within experimental error with those of ΔF mentioned above. The temperature dependence of ΔF and ΔL was found to be nearly the same, both at 685 and at 735 nm. (3) Analogously to the proposal concerning the 0.15 μs luminescence in photosystem II (Sonneveld, A., Duysens, L.N.M. and Moerdijk, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5889–5893), we propose that recombination of the oxidized primary donor P-700⁺ and the reduced acceptor A^?, probably A^?₁, of PS I causes the observed fast luminescence. The effect of an external magnetic field on this emission may be explained by the radical pair mechanism. The field-induced increase of the 0.1–0.2 μs luminescence seems to be at least in large part responsible for the observed increase of the total (prompt + delayed) emission measured during continuous illumination in the presence of a magnetic field.     

Bacteriorhodopsin (BR570) bathochromic band shift in an external electric field

In dry films of bacteriorhodopsin-containing purple membranes from Halobacterium halobium the external electric field (10⁴–10⁵ V · cm^?1) induces the appearance of a product spectrally close to the initial intermediate of bacteriorhodopsin (BR) photochromic cycle (batho form, K). This result and also preliminary data of the electret-thermal analysis of the preparations suggest that the dielectric polarization in chromophore-protein-lipid complexes might be an essential step of the primary stabilization of light energy in photo-bioenergetic processes.     

Studies on the temperature effect on bacteriorhodopsin of purple and blue membrane by fluorescence and absorption spectroscopy

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 340 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of bRp.As temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp increases.The binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to bRp.These results suggest that significant structural difference existed between bRband bRp.It was also found that both kinds of bR are highly thermal stable.The maximum wavelength of theprotein fluorescence emission only shifted from 340 nm to 346 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at380 nm formed.The transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high temperature.This phenomenon has not been reported before.     

Fluorescence spectra,fluorescence quantum yield and dissociation constant of sarafloxacin

In this study, the fluorescence spectra of sarafloxacin (SAR) under different pH conditions were investigated to determine the structural changes due to protonation that result from change in pH. At pH < 1.02, SAR exists in the H₃L²⁺ form for which the maximum fluorescence emission wavelength was about 455 nm. At pH 1.87–4.94, SAR exists in the H₂L⁺ form in which H₃L²⁺ loses one proton in the nitrogen molecule at the 1‐position in the quinoline ring. Fluorescence intensity was strong and steady and the maximum emission wavelength was 458 nm. At pH 7.14–9.30, the maximum emission wavelengths were gradually blue shifted to 430 nm with increase in pH, here SAR exists in the form of a bipolar ion HL in which H₂L⁺ loses a carboxyl group proton. At pH > 11.6, HL transforms into anionic L^? in which HL loses one proton from the piperazine ring, leading to a decrease in fluorescence intensity, and the maximum emission wavelength was red shifted to approximately 466 nm. The two‐step dissociation constant pKa for SAR was calculated, pK _a1 was 6.06 ± 0.37 and pK _a2 for SAR was 10.53 ± 0.19. In a pH 3.62 buffer solution with quinine sulfate as the reference, the fluorescence quantum yield of SAR at the maximum excitation wavelength of 276 nm was 0.09.     

Synthesis and characterization of high quantum yield and oscillator strength 6‐chloro‐2‐(4‐cynophenyl)‐4‐phenyl quinoline (cl‐CN‐DPQ) organic phosphor for solid‐state lighting

A novel blue luminescent 6‐chloro‐2‐(4‐cynophenyl) substituted diphenyl quinoline (Cl‐CN DPQ) organic phosphor has been synthesized by the acid‐catalyzed Friedlander reaction and then characterized to confirm structural, optical and thermal properties. Structural properties of Cl‐CN‐DPQ were analyzed by Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) spectroscopy, X‐ray diffraction technique (XRD) and scanning electron microscopy (SEM) and energy dispersive analysis of X‐ray (EDAX) spectroscopy. FTIR spectra confirmed the presence of different functional groups and bond stretching. ¹H–NMR and ¹³C–NMR confirmed the formation of an organic Cl‐CN‐DPQ compound. X‐ray diffraction study provided its crystalline nature. The surface morphology of Cl‐CN‐DPQ was analyzed by SEM, while EDAX spectroscopy revealed the elemental analysis. Differential thermal analysis (TGA/DTA) disclosed its thermal stability up to 250°C. The optical properties of Cl‐CN‐DPQ were investigated by UV–vis absorption and photoluminescence (PL) measurements. Cl‐CN‐DPQ exhibits intense blue emission at 434 nm in a solid‐state crystalline powder with CIE co‐ordinates (0.157, 0.027), when excited at 373 nm. Cl‐CN‐DPQ shows remarkable Stokes shift in the range 14800–5100 cm^?1, which is the characteristic feature of intense light emission. A narrow full width at half‐maximum (FWHM) value of PL spectra in the range 42–48 nm was observed. Oscillator strength, energy band gap, quantum yield, and fluorescence energy yield were also examined using UV–vis absorption and photoluminescence spectra. These results prove its applications towards developing organic luminescence devices and displays, organic phosphor‐based solar cells and displays, organic lasers, chemical sensors and many more.     

Effects of electric field on the photocycle of bacteriorhodopsin

The photoconversion of bacteriorhodopsin and the effects of an applied electric field (5 · 10⁷ V · m^?1) were studied in dry films of purple membranes from Halobacterium halobium. The electric field was found to cause at least two different effects: (1) it blocks in part the formation of the batho-bacteriorhodopsin (K), most probably due to electrically-induced dark transition of some bacteriorhodopsin molecules into the photochemically inactive form; (2) it decreases the rate of the intermediate M decay, the rise time of the M formation being unaffected by electric field. The observed phenomena may suggest a feedback control mechanism for the regulation of the bacteriorhodopsin photocycle in purple membranes.     

Flash photometric experiments on the photochemical cycle of bacteriorhodopsin

The photochemical reaction cycle of bacteriorhodopsin was investigated by means of flash photometric methods. Three different intermediates with absorption maxima at about 630 nm, 411 nm, and 646 nm could be detected. Kinetic data of the occurrence of these intermediates were obtained from isolated purple membrane in different mediums and from intact halobacteria. An activation energy of 14.1±0.4 kcal·mol⁻¹ and of about 19 kcal·mol⁻¹ for the formation of bacteriorhodopsin 411 and of bacteriorhodopsin 565, resp., was calculated. pH-changes in the medium caused by the reaction cycle of bacteriorhodopsin were detected by use of the pH-indicator bromocresol green.