CHLOROPHYLL a AND CAROTENOID AGGREGATES AND ENERGY MIGRATION IN MONOLAYERS AND THIN FILMS

Abstract— The spectra of absorption, fluorescence and excitation of monolayers and thin films containing chlorophyll a together with a carotenoid (cis-β-carotene, trans-β-carotene, fucoxanthin, or zeaxanthin), were measured at — 196°C. The concentration ratios used, (Chl)/(Car), were 6:1, 4:1, 3:1, 2:1, 1:1 and 1:3, and the area densities, 3·70, 2·55, 1·76, 0·71, 0·37 and 0·17 nm²/pigment molecule. In dilute monolayers, (3·70 nm²/molecule), with a constant concentration ratio (Chl)/(Car) = 3:1, evidence of three β-carotene forms, with absorption bands at 460, 500 and 520 nm (C₄₆₀, C₅₀₀ and C₅₂₀), and of a chlorophyll a form with an absorption band at 669–672 (Chl_669–672) was found. On increasing the density to 0·2–0·3 nm²/molecule, a conversion of C₄₆₀ and C₅₂₀ into C₅₀₀, was observed, and several more additional (probably more strongly aggregated) chlorophyll a forms appeared, with absorption bands at 672–733 nm. With excess carotene [(Chi)/(Car) = 1:3] the forms C₄₆₀, C₅₀₀, C₅₂₀ and Chl_669–672 were present even in the most dense films (0·2–0·3 nm²/molecule). The same was found with other carotenoids: if one of the pigments was in excess, aggregated forms of the other tended to disappear. In the transfer of energy from carotenoids to chlorophyll a, C₅₀₀ was found to be the main donor. In layers with a concentration ratio (Chl)/(Car) = 3:1, the efficiency of transfer was less than 10 per cent at the lowest density used (3·70 nm²/molecule); it increased to 50 per cent, as the density was increased to 0·20 nm²/molecule. When the relative concentration of the carotenoid was increased to (Chl)/(Car) = 1:1, the efficiency of energy transfer dropped to 25 per cent even at 0·20 nm²/molecule. It seems that the efficiency of energy transfer between carotene molecules (prior to its transfer to chlorophyll a) is low, and effective transfer occurs only between β-carotene and immediately adjacent chlorophyll a molecules.     

The light-dependent oxygen reduction by monolayers of hydrated chlorophyll a oligomer

The phenomenon of light-dependent O₂ uptake by monolayers of hydrated chlorophyll a oligomer deposited by the Langmuir-Blodgett technique on an SnO₂ optically transparent electrode has been observed. Spectra of cathodic photocurrents coincided with the absorption spectrum of hydrated oligomer of chlorophyll a. In the presence of an artificial electron donor, hydroquinone, and an oxygen electron acceptor, both the cathodic and the anodic photocurrent caused by dry and wet chlorophyll a molecules of monolayer were measured under illumination in the range 400–800 nm. The effects of electrode potentials and redox reagents on the magnitude of solar energy conversion by the chlorophyll a monolayer at the optically transparent electrode are discussed. ESR and circular dichroism spectra show that hydrated oligomer of chlorophyll a consists of six molecules of chlorophyll a bonded with water molecules.     

SPECTRAL PROPERTIES OF CHLOROPHYLL a MONOLAYERS: MONOLAYERS OF CHLOROPHYLL a AND PHEOPHYTIN AT A GAS-WATER INTERFACE*

Abstract— Surface and spectral properties of chlorophyll a monolayers were studied at a nitrogen-water interface. Direct spectral analysis of Chl monolayers indicated that compression results in a heterogenous mixture of Chl species. Fourth derivative and difference spectra showed the presence of minor bands at 692, 726 and 748 nm. The state of compression determines the quantity and type of spectral species formed. A Chl monolayer on an acid subphase results in the formation of a long wavelength absorbing species (705 nm) similar to that of pheophytin. The half-band width, optical density/monolayer, and extinction coefficients of Chl monolayers are given. It is concluded that in the monolayer the formation of various aggregated species of Chl can be induced.     

Neutral Nickel(II) and Copper(II) Tetraazamacrocyclic Complexes as Molecular Rods Attached to Gold Electrodes

New dithiolated derivatives of neutral Cu^II and Ni^II tetraazamacrocyclic complexes have been synthesized and characterized by spectroscopic and diffractional methods. These rod‐shaped molecules were assembled in monocomponent and mixed monolayers on gold electrodes. In the mixed monolayers, the active molecules were embedded in a hexanethiol matrix. The dithiolated complexes are oriented perpendicularly to the electrode, and reveal faster kinetics of electron transfer than those assembled in a single‐component monolayer. They appear as protrusions, which are easily addressed by using the STM method. In the presence of a suitable electron acceptor in the solution, the donor properties of the anchored Cu complex were weakened, which revealed donor–acceptor interactions with the monolayer. The peak position in the voltammogram indicates a stronger interaction of the solution‐based acceptor with the reduced Cu^II form than with the Cu^III complex. This suggests the possibility of switching the association on or off by applying an appropriate potential.     

FAR-INFRARED SPECTRA OF LANGMUIR-BLODGETT FILMS OF CHLOROPHYLL a,CHLOROPHYLL b,PHEOPHYTIN a and THEIR ADDUCTS WITH WATER and DIOXANE*

Fourier transform infrared spectra in the low frequency region (500–150cm^?1) of Langmuir-Blodgett films of chlorophyll a (Chi a), chlorophyll b (Chi b) and pheophytin a have been studied. Correlations between spectral changes in monolayer and multilayers of Chi a and Chi b and their adducts with water and dioxane have been established. Spectroscopic evidence has indicated that, although there are no individual absorption bands that can be assigned to pure Mg-nitrogen and/or Mg-oxygen stretching or bending modes, there are several bands in the400–200 cm^?1 region of the spectra containing considerable contributions from metal-nitrogen and metal-oxygen vibrational modes. These specific vibrations exhibit marked intensity changes and shifts upon water and dioxane interaction. The different states of chlorophyll aggregation in Langmuir-Blodgett mono- and multilayers films resulted in noticeable changes in their far-IR spectra.     

Determination of porphyrin dimer in a mixed monolayer of porphyrin/phospholipid transferred on ITO electrodes

The Langmuir–Blodgett method has been used to transfer mixed monolayers of a porphyrin (TMPyP) and a phospholipid (DMPA) from the air|water interface onto optically transparent indium–tin oxide (ITO) electrodes. The surface concentration of porphyrin, Γ, transferred on the ITO surface, has been obtained by integration of the reduction current from the cyclic voltammograms. The experimental Γ values ranged from Γ_m=5.19×10⁻¹¹ mol cm⁻², and corresponding to a compact monolayer of porphyrin monomers in a plane orientation with respect to the surface, to Γ_d=8.65×10⁻¹¹ mol cm⁻², and equivalent to the total amount of the porphyrin molecules at the air|water interface under a compression of 35 mN m⁻¹. Prior to the electrochemical experiments, the transmission spectrum was recorded. The surface concentration obtained of the porphyrin is not directly proportional to the transmission of the film, ΔT. This phenomenon is assigned to the dimer formation and, depending on the surface activity of the ITO electrodes, toward porphyrin adsorption. The dimer structure of TMPyP on an intact ITO electrode is altered with respect to that found at the air|water interface. A simple model has been developed to evaluate the contribution of monomer and dimer phases of the porphyrin in the mixed monolayer. Furthermore, spectroscopic measurements with linearly polarized light under oblique incidence have been performed in order to infer the plane orientation of the TMPyP molecules with respect to the ITO surfaces.     

Glycoprotein adsorption into bilirubin/cholesterol mixed monolayers at the air-water interface

The adsorption of α₁-acid glycoprotein into bilirubin/cholesterol mixed monolayers with various component molar ratios is investigated using surface pressure-area (π-A) isotherms and (dπ/dA)-A curves. The results showed that the surface area per molecule increased after the adsorption/insertion of glycoprotein molecules into the monolayers. The compressibility of mixed monolayers increased as a result of hydrogen bonding between bilirubin and glycoprotein molecules, while the interactions between bilirubin and cholesterol are weakened. The adsorption of glycoprotein into a monolayer induced changes in molecular surface area depending on the surface pressure and molar fraction of bilirubin. The transmission electron microscopy of mixed monolayers confirmed the insertion of glycoprotein particles of spherical shape with an average diameter of about 80 nm into the monolayer. The text was submitted by the authors in English.     

Molecular Recognition by Cyclophane/Guanidinium Supramolecular Receptor Embedded at the Air-Water Interface

In order to develop a supramolecular receptor through a self-assembling process, a site-specific host and an inclusion-type host were mixed as a Langmuir monolayer, and guest binding and pressure-induced fluorescence emission were investigated. A guanidinium amphiphile and several cyclophanes carrying hydrophobic moieties were used as the host molecules; molecular recognition of an aqueous fluorescent guest, 6-p-toluidino-2-naphthalenesulfonic acid (TNS) by binary mixed receptor monolayers was evaluated by a surface pressure-molecular area (π-A) isotherm and a surface fluorescence measurement. An apparent increase in fluorescence intensity was observed when the mixed monolayers of the guanidinium and cyclophane amphiphiles were compressed on an aqueous TNS solution. In contrast, single-component monolayers of the guanidinium or the cyclophane did not show a significant increase in fluorescence emission. In the mixed monolayers, the guest TNS would be bound to the interface by strong electrostatic interaction with the guanidinium, and inclusion of the formed complex probably suppresses the quenching effect in polar medium and/or self-quenching. Experiments with various mixing ratios of these components suggest selective formation of an equimolar cooperative receptor of the guanidinium and the cyclophane. Investigation of the cyclophane structures by fluorescence emission and a competitive binding experiment with another guest were also carried out.     

卟啉-噁二唑二元体系光诱导能量及电子传递研究

以5-对氨基苯基-10,15,20-三苯基卟啉及2-苯基-5-(对氨基苯基)-1,3,4-噁二唑为原料合成了系列卟啉-噁二唑二元化合物, 其结构通过¹H NMR, ESI-MS, IR, UV-Vis确定. 对合成化合物进行光谱性能测定, 结果表明, 在卟啉与噁二唑混合体系中, 存在着卟啉激发态分子向噁二唑基态分子的分子间电子传递过程, 导致卟啉激发态的荧光猝灭; 在卟啉-噁二唑二元体系中, 315 nm激发下发生了由激发态噁二唑基团至卟啉基团的能量传递, 导致噁二唑基团荧光猝灭, 卟啉基团荧光增强. 420 nm激发下不存在分子内卟啉基团向噁二唑基团的电子回传竞争; 电化学性能测定进一步表明从噁二唑基团向卟啉基团的电子传递是可能的. 因此卟啉-噁二唑二元化合物可能作为一种模型, 模拟光合作用中电子给体至叶绿素之间的电子传递过程.     

CHLOROPHYLL a FLUORESCENCE OF GONYAULAX POLYEDRA GROWN ON A LIGHT-DARK CYCLE AND AFTER TRANSFER TO CONSTANT LIGHT

Abstract— The chlorophyll a fluorescence properties of Gonyaulax polyedra cells before and after transfer from a lightdark cycle (LD) to constant dim light (LL) were investigated. The latter display a faster fluorescence transient from the level ‘I’ (intermediary peak) to ‘D’ (dip) to ‘P’ (peak) than the former (3 s as compared to 10 s), and a different pattern of decline in fluorescence from ‘I’ to ‘D’ and from ‘P’ to the steady state level with no clearly separable second wave of slow fluorescence change, referred to as ‘s' (quasi steady state)→‘M’ (maximum) →‘T’ (terminal steady state). The above differences are constant features of cells in LD and LL, and are not dependent on the time of day. They are interpreted as evidence for a greater ratio of photosystem II/photosystem I activity in cells in LL. After an initial photoadaptive response following transfer from LD to LL, the cell absorbance at room temperature and fluorescence emission spectra at 77 K for cells in LL and LD are comparable. The major emission peak is at 685–688 nm (from an antenna Chl a 680, perhaps Chl a-c complex), but, unlike higher plants and other algae, the emission bands at 696–698 nm (from Chl a_II complex, Chl a 685, close to reaction center II) and 710–720 nm (from Chl a₁, complexes, Chl a 695, close to reaction center I) are very minor and could be observed only in the fluorescence emission difference spectra of LL minus LD cells and in the ratio spectra of DCMU-treated to non-treated cells. Comparison of emission spectra of cells in LL and LD suggested that, in LL, there is a slightly greater net excitation energy transfer from the light-harvesting peridinin-Chl a (Chl a 670) complex, fluorescing at 675 nm, to the other antenna chlorophyll a complex fluorescing at 685–688 nm, and from the Chl a., complex to the reaction center II. Comparison of excitation spectra of fluorescence of LL and LD cells, in the presence of DCMU, confirmed that cells in LL transfer energy more extensively from the peridinin-Chl a complex to other Chl a complexes than do cells in LD.     

FLUORESCENCE SPECTROSCOPY OF A P700-CHLOROPHYLL-PROTEIN COMPLEX*

Abstract. Chlorophyll-protein complexes enriched in the Photosystem I reaction center chlorophyll (P700) exhibit a fluorescence emission maximum at 696 nm at - 196°C The height of this 696 nm emission relative to the emission at 683 nm from antenna chlorophyll a increases proportionally with the P700 concentration while the total fluorescence yield of the complex decreases. The 696 nm emission could possibly be from an absorbing form of antenna chlorophyll a that may be somewhat enriched along with P700 in Photosystem I fractions. However, evidence resulting from glycerol treatment which appears to decrease the rate of resonance energy transfer between antenna chlorophyll and P700 favors the hypothesis that the emission comes from a photooxidized P700 dimer (Chl⁺-Chl) absorbing near 690 nm. In turn, this fluorescence evidence provides additional support for the model of a P700 dimer involving exciton interaction. Absorption in the wavelength region of 450 nm specifically excites emission at 696 nm from the P700-chlorophyll complex.     

Compression of Self‐Assembled Nano‐Objects: 2D/3D Transitions in Films of (Perfluoroalkyl)Alkanes—Persistence of an Organized Array of Surface Micelles

Understanding and controlling the molecular organization of amphiphilic molecules at interfaces is essential for materials and biological sciences. When spread on water, the model amphiphiles constituted by C_nF_2n+1C_mH_2m+1 (FnHm) diblocks spontaneously self‐assemble into surface hemimicelles. Therefore, compression of monolayers of FnHm diblocks is actually a compression of nanometric objects. Langmuir films of F8H16, F8H18, F8H20, and F10H16 can actually be compressed far beyond the “collapse” of their monolayers at ～30 Ų. For molecular areas A between 30 and 10 Ų, a partially reversible, 2D/3D transition occurs between a monolayer of surface micelles and a multilayer that coexist on a large plateau. For A<10 Ų, surface pressure increases again, reaching up to ～48 mN m^?1 before the film eventually collapses. Brewster angle microscopy and AFM indicate a several‐fold increase in film thickness when scanning through the 2D/3D coexistence plateau. Compression beyond the plateau leads to a further increase in film thickness and, eventually, to film disruption. Reversibility was assessed by using compression–expansion cycles. AFM of F8H20 films shows that the initial monolayer of micelles is progressively covered by one (and eventually two) bilayers, which leads to a hitherto unknown organized composite arrangement. Compression of films of the more rigid F10H16 results in crystalline‐like inflorescences. For both diblocks, a hexagonal array of surface micelles is consistently seen, even when the 3D structures eventually disrupt, which means that this monolayer persists throughout the compression experiments. Two examples of pressure‐driven transformations of films of self‐assembled objects are thus provided. These observations further illustrate the powerful self‐assembling capacity of perfluoroalkyl chains.     

Lipolysis of didecanoyl-lecithin/triolein mixed monolayers by phospholipaseA 2

A study of enzyme lipolysis by pancreatic phospholipaseA ₂ and by vipera berus phospholipaseA ₂ on monomolecular mixed films of didecanoyl-lecithin and triolein on an aqueous subphase of pH 8 has been carried out. The influence of the composition of the mixed film, the surface pressure of the film and the amount and type of the injected enzyme on the lipolysis rate were studied.In order to relate the lipolytic activity with the monolayer state, the compression isotherms of the didecanoyl-lecithin/triolein mixed monolayers have also been obtained.The resuls are compared to observations on lipolytic activity of phospholipaseA ₂ on the didecanoyl-lecithin/cholesterol mixed monolayers. Triolein improves the kinetic conditions of the lipolysis of lecithin films in a higher degree than cholesterol. Probably it increases the enzyme penetration by the fluidifying effect exerted on the lecithin monolayers.     

Study on surface acid-base property of carboxylic acid-terminated self-assembled monolayers by cyclic voltammetry and electrochemical impedance spectroscopy

Cyclic voltammetry and electrochemical impedance spec-troscopy were used to study the surface acid-base property of carboxylic acid-terminated self-assembled monolayers(SAMs).A carboxylic acid-terminated thiol,such as thioctic acid(1,2-dithiolane-3-pentanoic acid),was self-assembled on gold electrodes.Electron transfer between the bulk solution and the SAM modified electrode was studied at different pH using Fe(CN)63-as a probe.The surface pka of thioctic acid was determined by cyclic voltammetry and electrochemical impedance spectroscopy to be 5.6 ±0.1 and 5.8±0.1,respectively.The method is compared with other methods of monolayer pKa measurement.     

Syntheses of Chalcone‐Type Chlorophyll Derivatives Possessing a Bacteriochlorin,Chlorin or Porphyrin π‐System and Their Optical Properties

C3‐(Trans‐2‐arylethenyl)carbonylated chlorophyll derivatives possessing a bacteriochlorin or chlorin π‐system were synthesized by cross‐aldol (Claisen–Schmidt) condensation of methyl pyrobacteriopheophorbide‐a or 3‐acetyl‐3‐devinyl‐pyropheophorbide‐a bearing the C3‐acetyl group with p‐(un)substituted benzaldehydes under basic conditions. The corresponding porphyrin‐type chlorophyll derivatives were prepared by the oxidation (17,18‐didehydrogenation) of the chlorin‐type. Their Qy absorption and fluorescence emission maxima in dichloromethane correlated well with Hammett substituent constants of the p‐substituents. Several electron‐withdrawing p‐substituents suppressed the emission due to photoinduced electron transfer quenching in a molecule. The substitution sensitivities for their maxima and fluorescence quantum yields decreased in the order of bacteriochlorin‐, chlorin‐ and porphyrin‐type derivatives.     

FREE ENERGY DEPENDENCE OF THE QUENCHING OF CHLOROPHYLL a FLUORESCENCE BY SUBSTITUTED QUINONES*

The quenching of chlorophyll a (Chl a) fluorescence hy a series of substituted benzoquinones. naphthoquinones and anthraquinones has been examined employing ethanol and acetonitrile as solvents. All quinones are good quenchers of fluorescence. There is an excellent linear relation between the Stern-Volmer quenching constants, K, and the polarographic half wave potentials (E₁₂) of the quinones, with more oxidizing quinones being better quenchers. The quenching data are consistent with the excited state half wave potential of ?1.31 eV predicted theoretically, demonstrating that the kinetically estimated value of the Chl a excited state reduction potential agrees with that expected on spectroscopic grounds. The results of quenching are not in agreement with the conventional Marcus theory of electron-transfer reactions, as there is no evidence of quenching constant. K_q. decrease vsΔG⁰ even for free energy changes nearly twice that expected for the onset of the Marcus inverted region. However, the kinetically estimated K_q values are in good agreement with the ones calculated by using the Rehm and Weller equation for fluorescence quenching by electron transfer. Our experimental results support the electron transfer mechanism of quenching proposed by Seely.     

Properties of Spreaded Collagen I Monolayers on the Surface of Aqueous tert-Butanol and n-Hexanol Solutions

Properties of the monolayers of collagen isolated from the sclera of pig's eye are studied at the air–water interface with increasing tert-butanol or n-hexanol concentrations in a subphase. In the case of aqueous n-hexanol solutions, its adsorption on the subphase surface results in the formation of mixed monolayer whose properties depend on n-hexanol concentration in the subphase and the ratio between the number of alcohol and collagen molecules in the monolayer. At higher n-hexanol surface concentration, the phase separation of the monolayer into the domains of the condensed phase of alcohol and fibrous collagen occurs. A decrease in water activity in the presence of tert-butanol leads to a drastic reduction of collagen surface activity. This effect can be explained by both the constrained collagen spreading on the surface of tert-BuOH solutions and adsorption of alcohol molecules on collagen resulting in macromolecule hydrophilization. Alcohol critical concentrations are disclosed above which collagen monolayers are not formed.     

Fabrication and Visualization of Metal‐Ion Patterns on Glass by Dip‐Pen Nanolithography

Fluorescent self‐assembled monolayers (SAMs) are used as dip‐pen nanolithography (DPN) substrates for the fabrication of patterns of Ca²⁺ and Cu²⁺ ions. The driving force for the transfer of these ions from an atomic force microscopy (AFM) tip to the surface is their complexation to organic ligands on the monolayer. By means of fluorescent surfaces, the patterns can be visualized under a fluorescence microscope. We use a custom‐built atomic force fluorescence microscope (AFFM), a combination of atomic force and confocal fluorescence microscopes, to deposit the metal ions onto the sensing SAMs by DPN and to subsequently visualize modulations of fluorescence intensity in a sequential write–read mode.     

Monolayers of an amphiphilic para-carboxy-calix[4]arene act as templates for the crystallization of acetaminophen

The title compound, 5,11,17,23-tetra-carboxy-25,26,27,28-tetradodecyloxy-calix[4]arene, 1, has been studied at the air–water interface, self-assembled as Langmuir monolayers, for its ability to interact with an active pharmaceutical ingredient (API), acetaminophen (APAP), and to initiate its crystallization. The Π/A isotherm study shows that there is a clear interaction between 1 and APAP causing an expansion of the monolayer. In addition to the known phase transition occurring at a surface tension of 38 mN m^?1, an additional kink is observed in the compression isotherm for concentrations of APAP above 40 mM suggesting that this API is causing an additional phase transition of the monolayer. Interface-initiated crystallization studies show that the presence of a monolayer spread on a supersaturated solution of APAP (26 g L^?1) triggers this API crystal growth from the interface. The transfer of 1-based monolayers on glass surfaces has been carried out using the Langmuir–Blodgett technique. The so-produced monolayers have been shown to template the crystallization of APAP. LB films of 1 have characterized using imaging and spectroscopic ellipsometry. The results suggest that each monolayer has an average thickness of 18 Å, which is consistent with the molecular structure of 1 self-organized parallel to the interface with the alkyl chains pointing out parallel to the axis of the macrocycle and without interdigitation of the alkyl chains. The presence of APAP in the subphase during the LB transfer causes a limited but relevant increase in the layer thickness. The study of the capabilities of the LB films to initiate crystallization of APAP is also demonstrated showing the influence of the monolayer packing on the quantity of formed crystals.     

RUTHENIUM RED INHIBITION OF OXYGEN EVOLUTION AND SPECIFIC RELEASE OF THE EXTRINSIC 16 kDa POLYPEPTIDE IN A PHOTOSYSTEM II PREPARATION

The inhibitory effect of the dye ruthenium red was studied in photosystem II-enriched submembrane fractions. A number of distinct types of interaction were found, which differed in their concentration range and required incubation time. Ruthenium red instantaneously quenches the initial chlorophyll a fluorescence level (F₀) and the maximum fluorescence level (F_m) by enhancing radiationless deactivation in the chlorophyll light harvesting complex. Associated with this quenching of fluorescence is an instantaneous decrease in the quantum yield of oxygen evolution. Ruthenium red also inhibited the light saturated rate of oxygen evolution and the variable fluorescence, monitored 80 µs after a saturating excitation-flash. These inhibitions increased with incubation time and became greater than 50% within 5 min. Although ruthenium red was known to affect Ca²⁺ or Cl^? sites specifically, the inhibitory action was more pronounced than simple Ca²⁺ or Cl^? depletion. Incubation with ruthenium red for 5 min blocks the Z P680⁺ → Z⁺ P680 charge transfer reaction. Upon mixing with the photosystem II preparation, ruthenium red induced specific release of the extrinsic 16 kDa polypeptide associated with water-splitting without release of Mn. It is proposed that the inhibitor produces an ionic imbalance which alters the configuration of the donor side of photosystem II.