Effect of solvents,solvent mixture and silver nanoparticles on photophysical properties of a ketocyanine dye

The effect of solvents of varying polarity and hydrogen bonding ability, solvent mixture and silver nanoparticles on the photophysical properties of a ketocyanine dye, 2,5‐di[(E)‐1‐(4‐diethylaminophenyl) methylidine]‐1‐cyclopentanone (2,5‐DEAPMC), is investigated at room temperature. Solvent effect is analyzed using Lippert–Mataga bulk polarity function, Reichardt's microscopic solvent polarity parameter, and Kamlet's and Catalan's multiple linear regression approaches. The spectral properties better follow Reichardt's microscopic solvent polarity parameter than the Lippert–Mataga bulk polarity function. This indicates that both general and specific solute–solvent interactions are operative. Kamlet's and Catalan's multiple linear regression approaches indicate that polarizability/dipolarity solvent influences are greater than hydrogen bond donor and hydrogen bond acceptor solvent influences. The solvatochromic correlations are used to estimate excited state dipole moment using the experimentally determined ground state dipole moment. The excited state dipole moment of the dye is found to be larger than its corresponding ground state dipole moment and ground and excited state dipole moments are not parallel, but subtend an angle of 77°. The absorption and emission spectra are modulated in the presence silver nanoparticles. The fluorescence of 2,5‐DEAPMC is quenched by silver nanoparticles. The possible fluorescence quenching mechanisms are discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of solvents on photophysical properties and quenching of 2‐{[3‐(1H‐benzimidazole‐2‐yl) phenyl] carbonoimidoyl}phenol

The effect of solvents of varying polarity on the absorption and fluorescence emission of the Schiff base, 2‐{[3‐(1H‐benzimidazole‐2‐yl) phenyl]carbonoimidoyl}phenol, was studied using Lippert‐Mataga bulk polarity function, Reichardt's microscopic solvent polarity parameter and Kamlet's multiple linear regression approach. The spectral properties follow Reichardt's microscopic solvent polarity parameter better than Lippert‐Mataga bulk polarity parameter, indicating the presence of both general solute–solvent interactions and specific interactions. Catalan's multiple linear regression approach indicates the major role of solvent polarizability/dipolarity influence compared with solvent acidity or basicity. The solvatochromic effect was utilized to calculate the dipole moments of ground and excited states of the Schiff base using different methods. Bathochromic shift in the emission spectrum and the increase in dipole moment in the excited state signifies the intramolecular charge transfer character in the emitting singlet state. Fluorescence quenching by aniline was also studied in 1,4‐dioxane and n‐butanol, and the results were analyzed using sphere of action static quenching and finite sink approximation models. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of solvent polarity on the absorption and fluorescence spectra of chlorogenic acid and caffeic acid compounds: determination of the dipole moments

The effects of solvent polarity on absorption and fluorescence spectra of biologically active compounds (chlorogenic acid (CGA) and caffeic acids (CA)) have been investigated. In both spectra pronounced solvatochromic effects were observed with shift of emission peaks larger than the corresponding UV‐vis electronic absorption spectra. From solvatochromic theory the ground and excited‐state dipole moments were determined experimentally and theoretically. The differences between the excited and ground state dipole moment determined by Bakhshiev, Kawski–Chamma–Viallet and Reichardt equations are quite similar. The ground and excited‐state dipole moments were determined by theoretical quantum chemical calculation using density function theory (DFT) method (Gaussian 09) and were also similar to the experimental results. The HOMO‐LUMO energy band gaps for CGA and CFA were calculated and found to be 4.1119 and 1.8732 eV respectively. The results also indicated the CGA molecule is more stable than that of CFA. It was also observed that in both compounds the excited state possesses a higher dipole moment than that of the ground state. This confirms that the excited state of the hydroxycinnamic compounds is more polarized than that of the ground state and therefore is more sensitive to the solvent. Copyright © 2015 John Wiley & Sons, Ltd.     

A combined spectroscopic and TDDFT investigation of the solute‐solvent interactions of two coumarin derivatives

The UV/Vis absorption and fluorescence characteristics of 3‐cyano‐7‐hydroxycoumarin [ CHC ] and 7‐amino‐4‐methyl‐3‐coumarinylacetic acid [ AMCA‐H ] were studied at room temperature in several neat solvents and binary solvent mixtures of 1,4‐dioxane/acetonitrile. The effects of solvent on the spectral properties are analyzed using single and multi‐parameter solvent polarity scales. Both general solute/solvent interactions and hydrogen bond interactions are operative in these systems. The solvation of CHC and AMCA‐H dyes in 1,4‐dioxane/acetonitrile solvent mixtures has been studied. The solutes CHC and AMCA‐H are preferentially solvated by acetonitrile and a synergistic effect is observed for both molecules in dioxane/acetonitrile solvent mixtures. In addition, using the solvatochromic method the ground‐ and the excited‐state dipole moments of both the dyes were calculated. The ground‐ and excited‐state dipole moments, absorption and emission maxima and HOMO–LUMO gap were also estimated theoretically using B3LYP/6–311+ G (d,p) level of theory in the gaseous phase, dioxane and acetonitrile solvents. Furthermore, changes in dipole moment values were also calculated using the variation of Stokes shift with the molecular–microscopic empirical solvent polarity parameter ( ). The observed excited‐state dipole moments are larger than their ground‐state counterparts, indicating a substantial redistribution of the electron densities in a more dipolar excited state for both coumarins investigated.     

Numerical studies of the membrane fluorescent dyes dynamics in ground and excited states

Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10 ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.     

Quantum chemical computations and photophysical spectral features studies of two coumarin compounds

An attempt was made to determine the ground state and excited state dipole moments and quantum chemical computations of two coumarin compounds, namely 3‐hydroxy‐3‐[2‐oxo‐2‐(2‐oxo‐2H‐chromen‐3‐yl)‐ethyl]‐1,3‐dihydro‐indol‐2‐one (3HOCE) and 3‐[2‐(8‐methoxy‐2‐oxo‐2H‐chromen‐3‐yl)‐2‐oxo‐ethylidene]‐1,3‐dihydro‐indol‐2‐one (3MOCE). Both compounds displayed a red shift with enhancement in solvent polarity. The larger excited state dipole moment indicated the more polar nature of the selected compounds in the excited state than in the ground state. Kinetic stability and chemical reactivity of the selected compounds were studied with help of the quantum chemical properties of the compounds such as frontier molecular orbital analysis using density functional theory calculations with B3LYP/6–311+G (d, p) basis sets. Molecular electrostatic potential, Mulliken charges, natural bond orbital, and nonlinear optical properties were further studied. NBO analysis showed proton transfer within the selected donor–acceptor, depicting the large energy of stabilization for the compounds. The calculated Fukui function inferred the local softness and electrophilicity indices of used solute compounds.     

Solvatochromic study of 3‐N‐(N′‐methylacetamidino)benzanthrone and its interaction with dopamine by the fluorescence quenching mechanism

The change in photophysical properties of the organic molecule due to solvatochromic effect caused by different solvent environments at room temperature gives information about the dipole moments of 3‐N‐(N′‐methylacetamidino)benzanthrone (3‐MAB). The quantum yield, fluorescence lifetime of 3‐MAB was measured in different solvents to calculate radiative and non‐radiative rate constants. The results revealed that the excited state dipole moment (μ_e) is relatively larger compared to the ground state dipole moment (μ_g), indicating the excited state of the dye under study is more polar than the ground state and the same trend is noticed with theoretical calculations performed using the CAM‐B3LYP/6‐311+G(d,p) method. Further, the study on preferential solvation was carried out for 3‐MAB dye in ethyl acetate–methanol solvent mixture. The fluorescence quenching method has been employed for the detection of dopamine using 3‐MAB as fluorescent probe, using steady‐state and time resolved methods at room temperature. The method enables dopamine in the micro molar range to be detected. Also, an attempt to verify the quenching process by employing different models has been tried. Various rate parameters are measured using these models, our results indicates the quenching process is diffusion limited.     

Specific interactions of alcohols and non‐alcohols with a biologically active boronic acid derivative: a spectroscopic study

The photophysical properties of 4‐fluoro‐2‐methoxyphenyl boronic acid (4FMPBA) are characterized using absorption and fluorescence techniques in series of non‐alcohols and alcohols. The results are analyzed using different solvent polarity functions and Kamlet and Catalan's multiple regression approaches. The excited state dipole moment and change in dipole moment are calculated using both the solvatochromic shift method and Reichardt's microscopic solvent polarity parameter . The ground state dipole moment is evaluated using quantum chemical calculations. It is found that general solute–solvent and hydrogen bond interactions are operative in this system. A red shift of ~ 9 nm in the emission spectra is observed with an increase in the solvent polarity, which depicts π→π^* transitions, as well as the possibility of an intramolecular charge transfer (ICT) character in the emitting singlet state of 4FMPBA. The relative quantum yield, radiative and non‐radiative decay constants are calculated in alkanes and alcohols using the single point method. It is found that the quantum yield of the molecule varies from 16.81% to 50.79% with the change in solvent polarity, indicating the dependence of fluorescence on the solvent environment. Copyright © 2015 John Wiley & Sons, Ltd.     

Ab initio calculations of molecular properties of low–lying electronic states of 2–cyclopenten–1–one – link with biological activity

Geometries, vibrational frequencies, vertical and adiabatic excitation energies, dipole moments and dipole polarizabilities of the ground and the three lowest electronic excited states, S(1)(n, π (*)), T(1)(n, π (*)), and T(2)(π, π (*)) of the 2-cyclopenten-1-one molecule (2CP) were calculated at the CCSD and CCSD(T) levels of approximation. Our results indicate that two triplets T(1)(n, π (*)) and T(2)(π, π (*)) are lying very close each to other, while the singlet S(1)(n, π (*)) is well above them. There are dramatic changes in dipole moments for (n, π (*)) excited states in respect to the ground state. On the other hand the T(2)(π, π (*)) state has a similar dipole moment as the ground state. These changes can be interpreted within the MO picture using electrostatic potential maps and changes in model IR spectra. Our CCSD(T) dipole moment data for the ground state and almost isoenergetic triplets T(1)(n, π (*)) and T(2)(π, π (*)) are 1.469?a.u., 0.551?a.u., and 1.124?a.u., respectively. Dipole polarizabilities of investigated excited states are much less affected by electron excitations than dipole moments. These are the first dipole moment and polarizability data of 2CP in the literature. The changes of molecular properties upon excitation to S(1)(n, π (*)) and T(1)(n, π (*)) correlate with the experimental data on the biological activity of 2CP related to the α, β-unsaturated carbonyl group.     

Pressure-induced phase transitions of lipid bilayers observed by fluorescent probes Prodan and Laurdan

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-dodecanoyl-2-(dimethylamino)naphthalene (Laurdan) in bilayer membranes of 1,2-distearoylphosphatidylcholine (DSPC) were observed as a function of pressure at constant temperature. The emission spectra of Prodan and Laurdan varied with the pressure-induced states of bilayer membranes. The maximum emission wavelength (lambda(max)) of Prodan characteristic of the liquid crystalline (L(alpha)), lamellar gel (L(beta)') and pressure-induced interdigitated gel (L(beta)I) phases of the DSPC bilayer was 480, 440 and 500 nm, respectively. On the other hand, the lambda(max) of Laurdan characteristic of the L(alpha) and L(beta)' phases was 480 and 440 nm in a similar manner as Prodan probe. However, no change in the lambda(max) was observed in spite of the occurrence of the interdigitation of bilayer. Since the lambda(max) reflects the solvent property around the probe molecules, we could speculate about the location of fluorescent probe in the bilayer membranes. In the L(alpha) phase the same chromophore group of Prodan and Laurdan probes distributes around phosphate group of lipid (i.e., polar region). The transformation of bilayer into the L(beta)' phase causes the Prodan and Laurdan molecules to move into the glycerol backbone (i.e., less polar) region. In the ripple gel (P(beta)') phase, the emission spectrum of Prodan shows a broad peak at about 480 nm and a shoulder around 440 nm, which means that the Prodan molecules are widespread over the wide range from the glycerol backbone to the hydrophilic part of bilayer. The P(beta)'/L(beta)I phase transition causes the Prodan molecule to squeeze out from the glycerol backbone region and to move the hydrophilic region near the bilayer surface. Contrarily, the Laurdan molecule was not squeezed out from the glycerol backbone region because the long acyl chain of Laurdan serves as an anchor in the hydrophobic core of bilayer. The ratio of fluorescence intensity of Prodan at 480 nm to that at 440 nm, F(480)/F(440), is available to observation of bilayer phase transitions. The plot of F(480)/F(440) versus pressure seems to be useful for the recognition of bilayer phase transition, especially the bilayer interdigitation.     

Intramolecular charge transfer processes in donor-acceptor substituted vinyltetrahydropyrenes.

Two novel donor-acceptor-substituted vinyltetrahydropyrene derivatives, 2-N,N-dimethylamino-7-(1-carbethoxyvinyl)-4,5,9,10-tetrahydropyrene, , and 2-N,N-dimethylamino-7-(1,1-dicyanovinyl)-4,5,9,10-tetrahydropyrene, , were synthesized and their photophysical properties investigated in solvents of different polarities. Our studies revealed the existence of intramolecular charge transfer excited states in these molecules. For both compounds the fluorescence maxima exhibited solvent polarity-dependent red shifts. These were quantitatively analysed by the Lippert-Mataga and Liptay equations to obtain the excited state dipole moments. Our results indicated that in the case of , emission takes place from a planar (1)CT state in all non-protic solvents. In the case of , the nature of the excited state depends on the solvent. A fast relaxation to a triplet state is proposed in cyclohexane. The emitting state in medium polar solvents is a planar (1)CT state. In highly polar solvents a twisted (1)CT state is invoked to explain the low fluorescence quantum yield. For both compounds CT nature of the emitting states were further confirmed by studies in acidic medium. The ground and excited state pK(a) values for and were determined using absorption and emission spectral changes observed in the presence of protic acids.     

Investigations on spectra and orientation of dipole moments of uracil and thymine by electrochromism

The direction of the ground stale dipole moment with respect to the transition moment to the lowest excited singlet state as well as the dipole moment in this excited state could be determined from the influence of an electric field on the light absorption of uracil and thymine in solution. By making use of results on the orientation of the transition moment in the molecular framework, the orientation of the ground-and excited-state dipole moments can also be fixed and compared with theoretical predictions. The agreement is fair. The measurements show that in both compounds a weak band is hidden under the longest-wavelength absorption band.     

Tunable fluorescence emission in pyrene-(2,2'-bipyridine) dyads containing phenylene-ethynylene bridges.

A synthetic route is described for the preparation of a series of pyrene-containing pi-conjugated 2,2'-bipyridine (bpy) ligands. These compounds have been investigated by steady-state and time-resolved fluorescence spectroscopy. They display intense visible absorption and fluorescence emission properties that can be very efficiently modulated by the complexation of zinc(ii) metal ions to the bpy coordinating unit. The solvatochromism of the emission band of the zinc(ii) complexes, the fluorescence quantum yields, and lifetimes in THF have been determined. Zinc(ii)-induced formation of a charge transfer singlet excited states induces an increase in dipole moment of more than 20 D. Semiempirical theoretical calculations were performed and allowed to assess the electronic nature of ground and excited states of the free ligands and and that of the corresponding zinc(ii) complexes.     

Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases.

Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, and its partition coefficient between the membrane and water depends on the phase state, i.e., on the packing of the bilayer. Prodan is sensitive to polarity variations occurring closer to the bilayer surface than those detected by Laurdan.     

A novel 4-(tetrahydro-2-furanmethoxy)-N-octadecyl-1,8-naphthalimide based blue emitting probe: Solvent effect on the photophysical properties and protein detection

A novel 4-(tetrahydro-2-mranmethoxy)-N-octadecyl-1,8-naphthalimide (4-TNI) blue emitting fluorophore was synthesized for solvent polarity probing and protein detection. The effect of various solvents on absorption and fluorescence spectra of 4-TNI was investigated. By comparison with other derivatives with heterocyclic electron-donating groups, 4-TNI had the advantage of higher fluorescence quantum yield. The bathochromic shift observed in absorption and fluorescence spectra of 4-TNI with increasing solvent polarity indicates that the transitions involved are ?? ?? ??*. The normalized transition energy value E _T ^N showed some scattering when plotted versus ????. According to the quantum mechanics second order perturbation method, the ground and excited state dipole moments of 4-TNI were calculated as 3.91 and 7.12 D, respectively. Density functional calculations were also used to obtain the ground and excited state dipole moments. The result was consistent with the experimental values. Binding of TNI with human serum albumin (HSA) was studied. Fluorescence data revealed that quenching of HSA fluorescence by 4-TNI was due to the formation of a 4-TNI-HSA complex. Hydrogen bonds and hydrophobic interactions are the driving force of complex formation. 4-TNI fluorescence was found to be very sensitive to quenching by HSA. Therefore, a new spectrofluorimetric method for detection of HSA in Tris-HCl buffer solution (pH 7.4) was developed. The linear range of the calibration curve was 0.1?C14.2 × 10^?6 M for HSA, with a detection limit (3??) of 1.37 × 10^?10 M. The method was applied to determination of total protein in clinical samples of human serum and the results were in good agreement with the data obtained by using Coomassie Brilliant Blue G-250 colorimetry.     

Comparative study of the photophysical properties of indoprofen photoproducts in relation with their DNA photosensitizing properties.

The photophysical properties of indoprofen photoproducts have been examined in various solvents by absorbance and emission spectroscopies in relation with their photosensitizing properties. The photophysical properties of 2-[4-(1-hydroxy)ethylphenyl]isoindolin-1-one (HOINP) and 2-(4-ethylphenyl)isoindolin-1-one (ETINP) are typical of a singlet excited state when the ones of 2-(4-acetylphenyl)isoindolin-1-one (KINP) are based on its triplet excited state according to previous work. The effect of solvent polarity on the absorption and fluorescence properties of HOINP and ETINP has been investigated as a function of Delta f, the Lippert solvent polarity parameter. A solvatochromic effect, function of the polarity region, has been observed for both photoproducts due to a change in the dipole moment of the compound upon excitation. In low-polarity regions, the excited state dipole moment of HOINP undergoes only a moderate increase (11.5 D) as compared to the dipole moment of the ground state (4.5 D) suggesting that the fluorescence arises from the locally excited state while in high-polarity regions it is strongly increased (42.9 D), which can imply that the emission takes place from a charge transfer state. In the case of ETINP, it would seem that the emitting state is rather a charge transfer state whatever the region is (16.9 and 31.8 D for the calculated excited-state dipole moments in the low and high-polarity regions, respectively). HOINP and ETINP do not produce thymine dimers by photosensitization but induce photooxidative damage via an electron transfer mechanism.     

Solvent polarity effect on excited-state lifetime of carotenoids and some dyes

The theory demonstrating the role of medium at the fluorescence quenching of polar compounds in solutions is briefly presented. It has been shown, that the rate of S(1) --> X(n) nonradiative conversion between the intramolecular charge transfer states depends on the permanent dipole moments in the ground (S(0)) and excited (S(1), X(n)) states as well as on solvent polarity. A relation for the rate of nonradiative excited-state energy conversion has been obtained and employed to test the known literature data for solvent effect on the S(1)-state lifetime of some biologically significant carotenoids and dyes (phthalimides). For phthalimides, the solvent isotope effect on the S(1)-state energy conversion, when hydrogen is replaced by deuterium in the OH groups of alcohols and water, has been analyzed. Based on the data for fluorescence quenching in solvents of different polarity, the dipole moments in the intermolecular charge transfer S(1) state have been obtained for carotenoids (peridinin, fucoxanthin, uriolide acetate) and for hydrogen-bonding complexes, which are formed by 4-amino-, 4-methylamino-, and 4-dimethylamino-N-methylphthalimides in alcohols and water.     

Effect of ethanol-induced lipid interdigitation on the membrane solubility of Prodan, Acdan, and Laurdan.

The effect of ethanol-induced lipid interdigitation on the partition coefficient (Kp) of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and its two derivatives, 6-acetyl-2-(dimethylamino)naphthalene (Acdan) and 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan), in L-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles has been examined by a precipitation method over the ethanol concentration range of 0-1.8 M. At 20 degrees C and in the absence of ethanol, the Kp values for Acdan, Prodan, and Laurdan are 2.0 x 10(3), 2.8 x 10(4), and 4.7 x 10(6), respectively. This result suggests that the Kp of Prodan and its derivatives is not simply a linear function of the polymethylene units. As DPPC undergoes the ethanol-induced phase transition from the noninterdigitated to the fully interdigitated gel state, Kp for Prodan and Acdan decreases by a factor of 5 and 2, respectively, whereas Kp for Laurdan exhibits no detectable changes with ethanol. The differences in Kp are in parallel with the differences in the fluorescence emission spectra of these probes over the ethanol concentration range examined. Previous fluorescence and infrared data indicated that membrane perturbation caused by the probes increases in the order: Laurdan > Prodan > Acdan. Thus, the degree of membrane perturbation also seems to be in parallel with Kp. Among these three probes, Prodan fluorescence reflects most correctly the ethanol-induced lipid interdigitation. In conclusion, the partitioning of small solutes in lipid membranes is significantly reduced by ethanol-induced lipid interdigitation, probably as a result of an increased membrane surface density due to the increased intramolecular lipid acyl chain ordering and a tighter overall intermolecular packing.     

4‐alkoxyethoxy‐N‐octadecyl‐1,8‐naphthalimide fluorescent sensor for human serum albumin and other major blood proteins: design,synthesis and solvent effect

A series of 4‐alkoxyethoxy‐N‐octadecyl‐1,8‐naphthalimides with intense blue fluorescence were designed and synthesized as polarity and spectrofluorimetric probes for the determination of proteins. In solvents of different polarities, the Stokes shifts of two dyes increased with increasing solvent polarity and fluorescence quantum yields decreased significantly, suggesting that electronic transiting from ground to excited states was π–π^* in character. Dipole moment changes were estimated from solvent‐dependent Stokes shift data using a solvatochromic method based on bulk solvent polarity functions and the microscopic solvent polarity parameter (). These results were generally consistent with semi‐empirical molecular orbital calculations and were found to be quite reliable based on the fact that the correlation of the solvatochromic Stokes shifts with was superior to that obtained using bulk solvent polarity functions. Fluorescence data revealed that the fluorescence quenching of human serum albumin (HSA) by dyes was the result of the formation of a Dye–HSA complex. The method was applied to the determination of total proteins (HSA + immunoglobulins) in human serum samples and results were in good agreement with those reported by the research institute. Copyright © 2012 John Wiley & Sons, Ltd.     

Polarized absorption spectra of green fluorescent protein single crystals: transition dipole moment directions

Polarized absorption spectra of orthorhombic crystals of wild-type green fluorescent protein (GFP) were measured between 350 and 520 nm to obtain information on the directions of the electronic transition dipole moments ((-->)m) of the chromophore relative to the molecular axes. The transition dipole moment orientation is a basic spectroscopic parameter of relevance to biologists when interpreting F?rster-type fluorescence resonance energy transfer data and for comparing absorbance and fluorescence spectra of GFP with quantum chemical calculations. Maximal extinction was obtained throughout the spectrum when the polarization direction of the electric vector of incident light was parallel to the c-axis of the crystal. The transition dipole moments were assumed to be parallel to the plane of the chromophore. With this assumption and the measured dichroic ratios in the crystals, the transition dipole moments associated with the neutral (lambda(max) = 398 nm) and anionic (lambda(max) = 478 nm) forms of the chromophore were found to subtend angles of approximately 26 degrees and 13 degrees (counterclockwise), respectively, with the vector that joins the phenolic and imidazolinone oxygen atoms of the chromophore.