LD Signal Research Articles

Secretory Proteins in the Skin Mucus of Nile Tilapia (Oreochromis niloticus) are Modulated Temporally by Photoperiod and Bacterial Endotoxin Cues

Although it is well known that the biological and physical characteristics of skin mucus in fishes are strongly affected by changes in environmental conditions, the influence of photoperiod and time-dependent bacterial endotoxin stimulation is not well documented. In the present study, we determined the diel variations in the basal activities of secretory proteins with known defense functions in the skin mucus of Nile tilapia (Oreochromis niloticus) maintained under two photic environments: equal length of day and night (12L:12D, LD) or total darkness (0L:24D, DD). A second experiment was conducted to determine how time-dependent (i.e., day versus night) lipopolysaccharide (LPS) challenge could influence these skin mucosal defenses. The results revealed that LD signal differentially modulated the activities of mucosal immune molecules. Fish subjected to LD regime showed significantly higher levels of skin mucus lysozyme and protease at nighttime than at daytime. This distinct feature was not observed in fish under DD. There was no general mucosal response patterns to time-dependent LPS challenge. Nonetheless, protease and lysozyme, which were identified to be at elevated levels at night, were significantly modulated when the endotoxin was administered at nighttime. Ceruloplasmin was the only molecule that responded to LPS challenge at daytime, where its activity significantly increased at 8 h post-stimulation. Collectively, the results revealed that photoperiod cues influenced the activities of mucosal defenses and this may play, at least in part, in the temporal sensitivity to bacterial endotoxin.

Gamma-quanta onboard identification in the GAMMA-400 experiment using the counting and triggers signals formation system.

GAMMA-400 (Gamma Astronomical Multifunctional Modular Apparatus) will be the new generation satellite gamma-observatory. Gamma-telescope GAMMA-400 consists of anticoincidence system (top and lateral sections - ACtop and AClat), the converter-tracker (C), time-of-flight system (2 sections S1 and S2), position-sensitive calorimeter CC1 makes of 2 strips layers and 2 layers of CsI(Tl) detectors, electromagnetic calorimeter CC2 composed of CsI(Tl) crystals, neutron detector ND, scintillation detectors of the calorimeter (S3 and S4) and lateral detectors of the calorimeter (LD). All detector systems ACtop, AClat, S1-S4, LD consist of two BC-408 based sensitive layers of 1 cm thickness each. Three apertures provide events registration both from upper and lateral directions. The main aperture provides the best angular (all strip layers information analysis) and energy (energy deposition in the all detectors studying) resolution. Gamma-telescope GAMMA-400 is optimized for the gamma-quanta and charged particles with energy 100 GeV detection with the best parameters in the main aperture. Triggers in the main aperture will be formed using information about particle direction provided by time of flight system and presence of charged particle or backsplash signal formed according to analysis of energy deposition in combination of both layers anticoincidence systems ACtop and AClat individual detectors. For double-layer ACtop taking into account both amplitude and temporal trigger marker onboard analysis only 2.8% photons will be wrongly recognized as electrons or protons for 100 GeV particles. The part of charged particles mistakenly identified as gammas is ∼10-5 using described algorithms. For E∼3 GeV less than 3% photons will be wrongly recognized as charged particles and fraction of wrongly identified charged particles will be also ∼10-5. In the additional aperture the particles identification is provided by analysis of signals corresponding to energy deposition in the individual detectors S2, S3 and fast signals from CC1 individual detectors discriminators. Low energy (0.2 - 10 MeV) photons in the lateral aperture recognizing by using simple anticoincidence signals from the individual detectors of LD. Gamma-quanta of higher energies are identified using energy deposition in the individual detectors of S3, S4, LD and fast signals from CC2 individual detectors discriminators. The results of anticoincidence system individual detectors thresholds modelling are discussed.

Non-intercalative binding mode of bridged binuclear chiral Ru(II) complexes to native duplex DNA

A pair of chiral binuclear ruthenium(II) complexes were prepared and their binding affinities towards double stranded native DNA were assessed by observing isotropic absorption, polarized light spectra – circular and linear dichroism (CD and LD), fluorescence quenching and DNA thermal denaturation. Upon binding to DNA, the complexes produced LD signals consisting of positive and negative signals in the absorption region, although they exhibited red shift and hypochromism in the absorption spectrum. These contrasting observations indicated that the binding modes of the complexes are largely deviated from classical intercalative binding. Groove binding of the complexes to DNA was found to be more likely than intercalative binding. The small increase of DNA melting temperature in the presence of the complexes indicated a predominance of DNA groove binding. The absence of “molecular light switch effect” further supported non-intercalative binding. The groove binding propensity of complexes was also supported by comparison of the resulting data with the [Ru(phen) 2(dppz)] 2+.

Nonlinear behavior during semi‐quantitative analysis of thin organic layers by laser desorption mass spectrometry

Characterization of the surface coverage and thickness of an organic thin film is particularly important in organic electronics and optoelectronics. For surface coverage down to the submonolayer level there is still a need for characterization methods which are easily applicable. In the present work we report on the evaluation of laser desorption mass spectrometry (LD-MS) for its use in thickness determination of organic thin films. Whereas LD-MS is well established as a soft ionization method for small molecules, its capability for use in quantitative analysis is nearly unexplored. We carried out experiments with two different molecules, 7,7,8,8-tetracyanoquinodimethane and hexabenzocoronene, in a series of experiments with increasing surface coverage. The obtained data were analyzed by plotting the LD signal intensities versus the relative layer thickness and they reveal a nonlinear behavior, which can be classified into regions of different desorption/ionization efficiencies. Visualization by atomic force microscopy reveals that the first efficiency change corresponds to the transition between incomplete and complete coverage of the metal surface by analyte molecules. A second transition is observed at high layer thickness where the signal intensity stays constant, independent of further thickness increments, and this is attributed to the limited penetration depth of the laser beam. The intermediate region between the two transitions shows a linear behavior and can thus be used for semi-quantitative thickness measurements. The efficiency change observed at the point of complete surface coverage is particularly useful for thin layer preparation of organic field effect transistors, where complete surface coverage is a minimum requirement.

Chiroptical Measurement of Chiral Aggregates at Liquid-Liquid Interface in Centrifugal Liquid Membrane Cell by Mueller Matrix and Conventional Circular Dichroism Methods

The centrifugal liquid membrane (CLM) cell has been utilized for chiroptical studies of liquid-liquid interfaces with a conventional circular dichroism (CD) spectropolarimeter. These studies required the characterization of optical properties of the rotating cylindrical CLM glass cell, which was used under the high speed rotation. In the present study, we have measured the circular and linear dichroism (CD and LD) spectra and the circular and linear birefringence (CB and LB) spectra of the CLM cell itself as well as those of porphyrine aggregates formed at the liquid-liquid interface in the CLM cell, applying Mueller matrix measurement method. From the results, it was confirmed that the CLM-CD spectra of the interfacial porphyrin aggregates observed by a conventional CD spectropolarimeter should be correct irrespective of LD and LB signals in the CLM cell.

DNA binding selectivity of oligopyridine-ruthenium(ii)-lysine conjugate

The synthesis, characterization and DNA binding properties of the complex [Ru(terpy)(4,4'-(COLysCONH(2))(2)bpy)Cl](3+) (1) have been studied. Complex (1) hydrolyzes to (2) with a calculated rate constant K(h) = 2.35 ± 0.08 × 10(-4) s(-1) and binds coordinatively to ct-DNA, with a saturation r-value at about 0.1. Stabilization of the ct-DNA helix at low electrolyte (NaClO(4)) concentration (10 mM) and destabilization at higher electrolyte concentrations (50-200 mM) was observed. Circular dichroism studies indicate that the hydrolyzed complex binds to DNA, increasing the unwinding of the DNA helix with an unwinding angle calculated as Φ = 12 ± 2°. The positive LD signal observed at 350 nm indicates some kind of specificity in complex orientation towards the global DNA axis. Complex (2) binds specifically to G4 on the central part of the oligonucleotide duplexes d(CGCGCG)(2) and d(GTCGAC)(2), as evidenced by NMR spectroscopy. Both lysine moieties were found to interact most likely electrostatically with the DNA phosphates, assisting the coordinative binding and increasing the DNA affinity of the complex. Photoinduced DNA cleavage by (2), upon UVA irradiation was observed, but despite its relative high DNA affinity, it was incomplete (~12%).

Effect of Number and Position of Positive Charges on the Stacking of Porphyrins along Poly[d(A-T)2] at High Binding Densities

At high porphyrin densities, the effects of the number and position of the positive charges of the periphery ring on the stacking of the porphyrin on poly[d(A-T)(2)] was investigated using polarized spectroscopy, including circular and linear dichroism (CD and LD, respectively). The CD spectrum of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin(TMPyP) consisted of two positive bands in the Soret absorption region at low [porphyrin]/[DNA base] ratios (R ratios) and changed to two distinguishable categories of the bisignate CD spectrum with increasing R ratio. These CD spectra were attributed to the monomeric groove binding, and the moderately and extensively stacked TMPyPs. In contrast, trans-bis(N-methylpyridinium-4-yl)porphyrin (trans-BMPyP) dominantly produced a CD spectrum that corresponded to the extensive stacking, except at the lowest R ratio that was used in this work (R = 0.04). However, for cis-bis(N-methylpyridinium-4-yl)porphyrin (cis-BMPyP), the intensity of the apparent bisignate CD signal was too small to assign it to the extensive stacking. Moreover, the shape of the CD spectrum in the DNA absorption region showed that the conformation of poly[d(A-T)(2)] was retained, in contrast to the extensively stacked TMPyP and trans-BMPyP. In the extensively stacked TMPyP- poly[d(A-T)(2)] assembly, the large negative LD signal in the Soret band was observed suggesting that the direction of the molecular planes of TMPyP was close to perpendicular with respect to the orientation axis (flow axis). In contrast, the LD spectrum of the trans-BMPyP-poly[d(A-T)(2)] complex produced positive LD signal in the same wavelength region, suggesting the orientation of the molecular plane was nearly parallel relative to the flow direction. Surprisingly, the LD signal in the DNA absorption region for both of the porphyrins was positive. Therefore, the helix axis of the DNA was near perpendicular relative to the flow direction in the porphyrin-polynucleotide assembly.

Dual functions of the human antimicrobial peptide LL-37—Target membrane perturbation and host cell cargo delivery

The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain ill-defined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced α-helix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel α-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.

Evaluation of a new high power, wide separation laser Doppler probe: Potential measurement of deeper tissue blood flow

Objective To compare the output from a novel high power, wide separation laser Doppler flow probe (DP1-V2-HP, 4 mm, with IRLD20) with that of a standard flow probe (DP1-V2, 0.5 mm, with DRT4) (Moor UK) and to explore its potential for use in the noninvasive measurement of blood flow in deeper tissues in humans. Methods Monte Carlo modeling was used to predict depths of light scattering in skin with each probe, geometry. Experimentally, forearm blood flow was measured at rest and during local warming of the skin surface and post occlusion reactive hyperaemia (PORH). Laser Doppler blood flux (LDF) and the power spectral density of its component frequency intervals, were compared. Results Monte Carlo modeling indicated that while the majority of wide probe LD signal derives from deeper tissue, a significant portion is from superficial (dermal) tissue (and vice versa for standard probe). Perturbation of local blood flow differentially increased LDF and spectral power as measured by the two probes, with the standard skin probe showing a significantly greater response to local skin warming ( p < 0.01). Conclusions These differences support our hypothesis that the wide probe is recording predominantly blood flux within the vasculature of sub-dermal tissue. This is in agreement with Monte Carlo simulation.

Intercalation of bulky Δ,Δ- and Λ,Λ-bis-Ru(II) complex between DNA base pairs

Interaction of Δ,Δ- and Λ,Λ-bis-Ru(II) complexes with native DNA was investigated by isotropic absorption and polarized spectroscopy including circular and linear dichroism (CD and LD). Despite the steric hindrance originating from its four bulky phenanthroline ligands at both ends of the molecule, this molecule rapidly intercalates between DNA base pairs. Intercalation was judged by large hypochromism and red shift in the UV–visible absorption spectra in the absorption region of the bridging moiety as well as in the metal-to-ligand-charge transfer absorption region. Further support for the intercalation is found in the fact that the magnitude of negative reduced LD signal in the absorption region of the bridging moiety was comparable to that of the DNA absorption region, indicating that the bridge connecting the two Ru(II) complexes is nearly parallel to the DNA base planes. No difference in the binding mode between the two enantiomers was observed. In the presence of either bis-Ru(II) complex, ethidium bromide, a classical intercalator, can intercalate into the empty sites but was not able to replace the Ru(II) complexes. Near the saturation, ground state interaction between ethidium and bis-Ru(II) complex was evident by LD.

Membrane Binding of pH-Sensitive Influenza Fusion Peptides. Positioning, Configuration, and Induced Leakage in a Lipid Vesicle Model

pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA2(1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion ( approximately 60-65 degrees relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue.

Supramolecular order following binding of the dichroic birefringent sulfonic dye Ponceau SS to collagen fibers

The optical anisotropies (linear dichroism or LD and birefringence) of crystalline aggregates of the sulfonic azo-dye Ponceau SS and of dye complexed with chicken tendon collagen fibers were investigated in order to assess their polarizing properties and similarity to liquid crystals. In some experiments, the staining was preceded by treatment with picric acid. Crystalline fibrous aggregates of the dye had a negative LD, and their electronic transitions were oriented perpendicular to the filamentary structures. The binding of Ponceau SS molecules to the collagen fibers altered the LD signal, with variations in the fiber orientation affecting the resulting dichroic ratios. The long axis of the rod-like dye molecule was assumed to be bound in register, parallel to the collagen fiber. Picric acid did not affect the oriented binding of the azo dye to collagen fibers. There were differences in the optical anisotropy of Ponceau SS-stained tendons from 21-day-old and 41-day-old chickens, indicating that Ponceau SS was able to distinguish between different ordered states of macromolecular aggregation in chicken tendon collagen fibers. In the presence of dichroic rod-like azo-dye molecules such as Ponceau SS, collagen also formed structures with a much higher degree of orientation. The presence of LD in the Ponceau SS-collagen complex even in unpolarized light indicated that this complex can act as a polarizer.

Stacking of meso-Tetrakis(3-N-methylpyridiniumyl)porphyrin on Poly[d(A-T)2]: Importance of the Distance between Porphyrin's Positive Charges

The meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (m-TMPyP)−poly[d(A-T)2] complex at a [porphyrin]/[DNA base] ratio of 0.05 was investigated by polarized spectroscopy, including circular and linear dichroism (CD and LD). The spectroscopic properties of the complex depend on the NaCl concentration and time, and can be classified into three types. At a NaCl concentration below 10 mM, a strong bisignate CD spectrum and negative LD spectrum in the Soret band were apparent immediately after mixing. As time lapsed, the spectral properties changed and the complex exhibited a small bisignate CD spectrum and a strong negative LD spectrum. On the other hand, at a NaCl concentration above 10 mM, the bisignate feature of the CD spectrum in the porphyrin absorption region exhibited little change. The LD signal in the DNA absorption region collapsed at this condition, while the CD signal was retained, indicating that DNA remained in the B-form but was compactly aggregated. Surprisingly, a strong positive band in the Soret region was apparent, and its magnitude increased with time. The observed spectral properties for the m-TMPyP−poly[d(A-T)2] complex are in contrast with those of the meso-tetrakis(4-N-methylpyridiniumyl)porphyrin−poly[d(A-T)2] complex, which binds at the groove of a polynucleotide at a low NaCl concentration and stacks at a high NaCl concentration. The difference in the binding mode may be attributed to the difference in distance between negatively charged phosphate groups: this distance is variable in the m-TMPyP molecule.

Invisible liposomes: refractive index matching with sucrose enables flow dichroism assessment of peptide orientation in lipid vesicle membrane.

Valuable information on protein-membrane organization may in principle be obtained from polarized-light absorption (linear dichroism, LD) measurement on shear-aligned lipid vesicle bilayers as model membranes. However, attempts to probe LD in the UV wavelength region (<250 nm) have so far failed because of strong polarized light scattering from the vesicles. Using sucrose to match the refractive index and suppress the light scattering of phosphatidylcholine vesicles, we have been able to detect LD bands also in the peptide-absorbing region (200-230 nm). The potential of refractive index matching in vesicle LD as a general method for studying membrane protein structure was investigated for the membrane pore-forming oligopeptide gramicidin incorporated into the liposome membranes. In the presence of sucrose, the LD signals arising from oriented tryptophan side chains as well as from n-->pi* and pi-->pi* transitions of the amide chromophore of the polypeptide backbone could be studied. The observation of a strongly negative LD for the first exciton transition ( approximately 204 nm) is consistent with a membrane-spanning orientation of two intertwined parallel gramicidin helices, as predicted by coupled-oscillator theory.

Seasonal variation in long-day stimulation of prolactin secretion in ewes.

Whereas ewes initiate reproductive activity in response to a photoperiod signal initiated after the winter solstice of 35 long days (35 LD) followed by short days, the reproductive axis fails to respond to this signal between the autumn equinox and the winter solstice. The aim of experiment 1 was to determine whether the prolactin axis, like the reproductive axis, is unresponsive to a 35 LD photoperiod signal followed by continuous exposure to short days between the autumn equinox and the winter solstice. Whereas the 35 LD signal from September 21 (</= 6 h increase in day length) failed to influence prolactin secretion, all other long-day treatments (> 6 h increase in day length) initiated a rise in prolactin in at least 75% of ewes in each group (p < 0.05). The aim of experiment 2 was to determine whether ewes failed to secrete prolactin during a 35 LD photoperiod from September 21 because they did not recognize a 6-h increase in day length at any time of year as a stimulatory photoperiod signal or because hypothalamic/pituitary regulation of prolactin synthesis or secretion is compromised in September. The results demonstrated that while hypothalamic regulation of prolactin secretion and pituitary stores of prolactin were normal at all times of year examined, the ability of ewes to secrete prolactin in response to a long-day photoperiod signal appears to be dependent on photoperiodic history rather than the time of year of the photoperiodic challenge.

Quercetin and DNA in solution: analysis of the dynamics of their interaction with a linear dichroism study

Flow-Linear Dichroism (LD) spectra on quercetin-DNA solutions (buffer-ethanol 30%) showed evidence that the flavonol can intercalate the biopolymer. There is no electrostatic component in the formation of the quercetin-DNA complex. The DNA concentration and the planarity of the chromophore are limiting factors in the interaction. There are no induced LD signals for concentrations of the biopolymer less than 7.8 × 10 −3M phosphate. The interaction is most probably of a hydrophobic nature between the most hydrophobic segment of the quercetin (benzopyran-4-one) and the intercalation site, which allows the chromophore to penetrate the DNA helix and to arrange its planar structure more or less parallel to the adjacent planes of the nitrogenous bases. A comparison between the planar and hydrophobic flavonol quercetin, and the non-planar and hydrophilic flavanone dihydroquercetin, showed that the interaction of the latter with DNA was strongly limited. The notable biological activity of the quercetin compared to the ‘weaker’ activity of the dihydroquercetin could also be derived from the different planarity (and probably hydrophobicity) of the two flavonoids. The very low concentration of the quercetin-DNA complex was efficiently shown by the high sensitivity of the LD technique, whereas it could not be resolved by isotropic UV-Vis and induced circular dichroism spectra. The hypothesis of a frame shift mutagenicity activity of quercetin ( Science 1977; 197: 577–578) is highly improbable. In fact, the affinity of quercetin for DNA, which emerges from this study, is very low compared with that of a typical intercalator agent ( Q. Rev. Biophys. I 1992; 25: 51–170)

Linear dichroism characteristics of ethidium–and proflavine–supercoiled DNA complexes

A flow linear dichroism technique is utilized to study the unwinding of supercoiled DNA induced by the binding of ethidium bromide (EB) and proflavine (PF) at different ratios r (drug added/DNA base). In the case of either EB or PF bound to linear calf thymus DNA, the reduced linear dichroism signals LD/A (LD: linear dichroism; A: absorbance, both measured at the same wavelength), determined at 258, and 520 or 462 nm (corresponding to contributions predominantly from the partially oriented DNA bases, intercalated EB, or PF, respectively) are nearly independent of drug concentration. In the case of supercoiled DNA, the magnitude of LD/A at 258 nm first increases to a maximum value near r = 0.04-0.05, and then decreases as r is increased further, mimicking the behavior of the sedimentation coefficients, viscosities, and gel electrophoresis patterns measured by other workers at similar values of r. However, LD/A at 520 nm, which is due to DNA-bound EB molecules, is constant within the range of r values of 0.02-0.06 in which the magnitude of LD/A determined at 258 nm due to the DNA bases exhibits a pronounced maximum. In contrast, in the case of PF, the magnitudes of LD/A determined at 258 or 462 nm are characterized by similar dependencies on r, both exhibiting pronounced maxima at r = 0.05; this parallel behavior is expected according to a simple intercalation model in which the DNA bases and drug molecules are stacked on top of one another, and in which both are oriented to similar extents in the flow gradient. The unexpected differences in the dependencies of (LD/A)258 and (LD/A)520 on r in the case of EB bound to supercoiled DNA, are attributed to differences in the net overall alignment of the EB molecules and DNA bases in the flow gradient. The magnitude of the LD signal at 258 nm reflects the overall degree of orientation of the supercoiled DNA molecules that, in turn, depends on their hydrodynamic shapes and sizes; the LD signals characterizing the bound EB molecules may reflect this orientation also, as well as the partial alignment of individual DNA segments containing bound EB molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Orientation of the pigments in Photosystem II: A low-temperature linear dichroism and polarized fluorescence emission study of chlorophyll-protein complexes isolated from Chlamydomonas reinhardtii

The orientation of the various absorbing and fluorescing dipoles in Photosystem II have been investigated by linearly polarized light spectroscopy at 5 K, performed on macroscopically oriented PS II complexes derived from Chlamydomonas reinhardtii. Linear dichroism and absorption spectra show that the Q Y transitions of the chlorophyll molecules are mostly tilted at less than 35° from the plane of largest cross-section of the particle (which in vivo coincides with the plane of the thylakoid membrane). The chlorophyll forms absorbing at 676 and 683 nm are oriented closer to the membrane than the forms absorbing at 665 and 670 nm which are tilted at approximately 35° from the plane. A dip observed around 680 nm in the LD/absorption spectra indicates a component tilted at a larger angle away from the membrane plane than the 676 nm- and 683 nm-absorbing species. A component weakly absorbing around 693 nm and exhibiting a negative LD (tilt larger than 35°) is clearly resolved. The amplitude of the LD at 693 nm relative to that observed at the maximum (676 nm) varies from sample to sample. In the blue spectral region, two populations of carotenoids are observed; one absorbs around 460 and 490 nm, while the other absorbs around 510 nm. They are oriented out of and near to the thylakoid plane, respectively. Comparison of polarized absorption and fluorescence spectra from the same oriented samples allows the assignment of the 695 nm fluorescence emission to the dipoles responsible for the LD signal at 693 nm.

GENETIC VARIATIONS AND LINOLENIC ACID INDUCED CHANGES IN THE ORIENTATION PATTERN OF CHLOROPHYLL a IN THYLAKOID MEMBRANES

Abstract Orientation pattern of the Qy absorption and emission dipoles of chlorophyll a were studied in wild type Scenedesmus obliquus and in mutants deficient in chlorophyll b and carotenoids. Fluorescence polarization ratio at –140°C and linear dichroism at 25°C were measured in whole cells and thylakoids aligned in polyacrylamide gel. Unlike normal thylakoids, mutants displayed fluorescence polarization ratios significantly lower than 1.0 and showed a negative LD signal around 672 nm, indicating the tendency of the Qy dipoles to tilt out from the membrane plane. Such an orientation pattern can also be artificially induced by treating normal thylakoids with linolenic acid.