Spectroscopic investigation of binding of three fluorescent nanomarkers to bionanomolecules of human serum albumin in dependence on pH

The analysis of fluorescent characteristics and degree of molecular association of three fluorescent nanomarkers (eosin, erythrosin and fluorescein) in solutions of human serum albumin (HSA) at different pHs is made. The common features for all three nanomarkers under influence of bionanomolecules of HSA are the quenching of the fluorescence, the red shift of maximum of fluorescence and the decrease of degree of molecular association for every fixed value of pH. The differences in dependences of fluorescence and degree of molecular association on pH between fluorescein and its halogen – derivatives (eosin and erythrosin) are registered. It is established that the quenching of fluorescence by HSA is of compound statically–dynamical type. The electronegativity of lateral atoms in structural formulas of nanomarkers forms the basis of explanation of all features of experimental data in the system “fluorescent nanomarker – protein – buffer solution”.     

Raman spectroscopy in investigations of secondary structure of human serum albumin at binding of nanomarkers of fluorescein family

The influence of binding of nanomarkers of fluorescein family to HSA on secondary structure of this protein at different values of pH was investigated by Raman spectroscopy method. The greatest changes in secondary structure of HSA, consisting in decreasing of α-helix sites, at binding of fluorescein to HSA occur at pH 5–6. The greatest changes in secondary structure of HSA, consisting in decreasing of α-helix sites, at binding of eosin or erythrosin to HSA take place at values of pH, smaller 5. The differences in changes in secondary structure of HSA at binding of these three nanomarkers are explained by dependences of binding of nanomarkers to HSA on pH which determined by value of electronegativity of atoms of lateral radicals in structural formulas of nanomarkers and, therefore, by value of pK of their ionized groups.     

Effect of proton-donor solvent and structural flexibility of prodan and laurdan molecules on their spectral-luminescent properties

The influence of structural flexibility on dipole moments, energy-level locations, and charge distributions in prodan and laurdan molecules was studied. A quantum-chemical calculation of isolated prodan and laurdan molecules in the fluorescent state geometry was conducted. Rate constants for radiative and non-radiative processes and fluorescence quantum yields for these probe molecules were calculated. Interaction centers of prodan and laurdan with a proton-donor solvent were estimated quantitatively. The possibility of using fluorescent probes for estimating the polarity of proton-donor and proton-acceptor solvents was shown. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 76, No. 3, pp. 334–341, May–June, 2009.     

Rotational Diffusion of Coumarins in Alcohols: A Dielectric Friction Study

The rotational dynamics of three structurally similar polar molecules viz., coumarin 440, coumarin 151 and coumarin 450 has been studied in alcohols at room temperature using steady-state fluorescence depolarization method and time correlated single photon counting technique. The observed reorientation times of all the three probes are found to be longer than those predicted by Stokes–Einstein–Debye (SED) hydrodynamic theory with stick boundary condition and a deviation towards super-stick behavior is noted. Dielectric friction theories of Nee–Zwanzig and van der Zwan–Hynes, which treat the solute as a point dipole, overestimate the dielectric friction contribution exhibited by all the three coumarins in alcohols. Results are discussed in the light of theoretical models and the possibility of hydrogen bonding between the amino group of the probe molecules and the hydroxyl group of the alcohols.     

Molecular association processes and fluorescent characteristics of nanomarkers of the fluorescein family in solutions of bovine serum albumin

An analysis of the molecular association and fluorescent characteristics of nanomarkers of the fluorescein family, viz., fluorescein, erythrosine, eosine, and Rose Bengal, in BSA solutions was conducted. For all the markers a decreasing degree of molecular association was observed in the BSA solutions as compared with the solutions without protein. In the solutions with BSA, fluorescence quenching and red shifting of the fluorescence spectrum maximum occurred for the solutions with BAS compared with solutions without protein for the markers of the fluorescein family. The dependences of the degree of molecular association on pH differed for fluorescein and its halogen derivatives. The efficiency dependences of nanomarker binding with BSA on pH differed for fluorescein and its halogen derivatives.     

Theory of polarized fluorescence of polyatomic radicals upon photodissociation of complex molecules in the gas phase

The effect of rotational predissociation on the polarization response of an ensemble of photofragments is investigated within the framework of the model with free scatter of fragments for the most general case when both original molecules and fragments are asymmetric rotators. Photoinduced decomposition reactions with single- and two-photon excitation of fragment fluorescence are analyzed. Second- and third-order orientational correlation functions determining the polarization degree of fragment fluorescence are calculated. The behavior of these correlation functions depending on the shape of original molecules and fragments, directions of dipole moments, and characteristic times of the process are analyzed. Institute of Molecular and Atomic Physics, National Academy of Sciences of Belarus, 70, F. Skorina Ave., Minsk, 220072, Belarus. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 65, No. 6, pp. 908–914, November–December, 1998.     

The effect of halogen substitution on the structure and electronic spectra of fluorone dyes

By means of the B3LYP density functional method with the use of the polarized continuum model PCM, we have performed quantum-chemical computations of the electronic absorption and fluorescence spectra of fluorone dianions: fluorescein, dibromofluorescein, eosin, erythrosine, and Rose Bengal in vacuum and methanol. We have revealed conformational features of the structure of fluorone dianions (charge redistribution, changes in the bond lengths and angles between bonds) second by the halogen substitution, the transition from the ground state to an excited state, and the change of the solvent (vacuum-methanol). Absorption and fluorescence wavelengths, constant dipole moments, transition dipole moments, and oscillator strengths have been calculated. We have showed that, upon halogenation of fluorones, the absorption spectra are redshifted and the Stokes shift decreases, which is qualitatively consistent with experimental results.     

Fluorescent characteristics of probes of the fluorescein family in human serum albumin solutions

Differences in human serum albumin solutions of fluorescein family probes in their molecular association, in the type of dependence of fluorescence on pH, and in fluorescence anisotropy have been shown. These are due to the electronegativities of the atoms of the lateral radicals in the structural formula and the pK values of the ionizable groups of the probes.     

Subnanosecond spectrofluorimetry of new indolocarbazole derivatives in solutions and protein complexes and their dipole moments

The spectral and temporal characteristics of new 6,12-dimethoxyindolo[3,2-b]carbazole, 5,11-dimethyl-6,12-dimethoxyindolo[3,2-b]carbazole, and 5,11-dihexyl-6,12-di(hexyloxy)indolo[3,2-b]carbazole fluorescence probes in organic solvents and protein complexes are studied. The dipole moments of indolocarbazoles in 1,4-dioxane were measured by electrooptical absorption method. The measured dipole moments have values within the range of (3.1–3.6) × 10⁻³⁰ C m in the equilibrium ground state and increase to (4.8–5.6) × 10⁻³⁰ C m after excitation. The excited state lifetime of indolocarbazole derivatives increases with increasing polarity and viscosity of the environment. The binding of indolocarbazoles with trypsinogen and human serum albumin increases the fluorescence intensity, changes the intensity ratio of fluorescence bands, and increases the average excited state lifetime of indolocarbazoles. The analysis of the instantaneous fluorescence spectra and fluorescence decay parameters at different wavelengths revealed the existence of several types of probe binding sites in proteins. It is found that the fluorescence characteristics of indolocarbazole derivatives depend on the conformation rearrangements of trypsinogen due to its thermal denaturation.     

Influence of temperature and ethanol content on aggregation of rhodamine 6G molecules in aqueous ethanol solutions

The effect of temperature on absorption spectra of Rhodamine 6G at concentrations of 10⁻⁶ and 10⁻³ M in water:ethanol solutions of various ethanol content was studied. The dimeric molecular fraction of Rhodamine 6G (10⁻³ M) was found as a function of temperature and ethanol content in the aqueous solution. It was shown that the absorption spectrum of Rhodamine 6G dimers (10⁻³ M) is dependent on temperature and ethanol content in the aqueous solution. Based on the relationship between J-and H-bands in the absorption spectrum of Rhodamine 6G dimers, both the angle between planes of associated Rhodamine 6G dye molecules and the free enthalpy of association were calculated. The structure of Rhodamine 6G dimers depends essentially on the ethanol content and aqueous ethanol solution temperature while experiencing the most temperature changes at an ethanol concentration of 25%. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 5, pp. 640–645, September–October, 2008.     

Efficiency of the structure analysis of complex molecules in the gas phase by their optical rotation

It was shown in the dipole approximation of optical rotation that in the general case only in orientationally anisotropic vapors is the rotational force dependent on the intramolecular orientation of both the electric and magnetic dipole moments. Expressions relating the optical rotational force to the intramolecular orientation of these moments, the orientational distribution in an anisotropic ensemble, and the configuration of a measurement have been obtained. Calculated dependences of the rotational force on the intramolecular orientation of the magnetic moment at a fixed electric moment and “rotational force excitation spectra” obtained for different types of rigid asymmetric top molecules and rotational contours are presented. It is proposed to measure the intramolecular orientation of the electric and magnetic dipole moments with the use of the rotational force normalized to that detected in the case of observation at a “magic” angle to the direction of the exciting light electric vector. Institute of Molecular and Atomic Physics of the Academy of Sciences of Belarus, 70, F. Skorina Ave., Minsk, 220072, Belarus. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 65, No. 6, pp. 843–849, November–December, 1998.     

Quenching of fluorescence of porphyrin molecules in their association in binary solutions

It has been established that the absorption and fluorescence spectra of octaethylporphin (OEP), mesotetraphenylporphin (TPhP), and its complex with zinc (Zn-TPhP) in glycerol doped with dimethylformamide (DMF) or ethanol differ from the analogous spectra of these compounds in ethanol or DMF, which is due to the formation of associates of porphyrin molecules in solutions of OEP, TPhP, and An-TPhP with glycerol. For TPhP and Zn-TPhP, these differences are small, the maxima of the bands are shifted toward longer wavelengths by 3–5 nm, and their halfwidths are increased by ∼ 16%; for OEP these changes are more significant. It is shown that the duration of fluorescence of associates is much shorter than that of monomers. The probable mechanisms of quenching of the fluorescence of dimers (oligomers) of molecules were considered. It has been suggested that one of the reasons for the increased rates of deactivation of the excited states of dimers (oligomers) may be the decreased energy interval between the excited and ground states of associates as compared with that of monomers. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 67, No. 1, pp. 21–25, January–February, 2000.     

The Orientation of the Phosphorescence Dipole Moment of Erythrosine B Within Its Molecular Frame

For an unequivocal interpretation of time-resolved phosphorescence depolarization measurements on dye molecules in biophysical systems, the orientation of the transition dipole moments within the frame of the dye has to be known. The goal of this work is to find the orientation of the phosphorescence dipole moment of erythrosine B within its molecular frame. To this end, we first determine four difference angles, exciting a molecule in two of its absorption bands and measuring the fluorescence and phosphorescence anisotropy for each excitation wavelength. The next step is to position the transition dipole moments within the molecular frame, combining the results of angle-resolved fluorescence depolarization measurements with the measured difference angles. It is shown that the phosphorescence dipole moment is tilted out of the plane of the molecule. Additionally, we find that the phosphorescence dipole moment is located above the fluorescence dipole moment and not above the absorption dipole moment. This induces an extra difference angle between the absorption and the phosphorescence dipole moments which in the past has been interpreted erroneously as a larger tilt out of the plane of the molecule.     

Determination of the directions of the transition dipoles in tetrabutylperylene in stretched polymers

The directions of the transition dipole moments of 2,5,8,11,-tetra-butylperylene were determined from angle-resolved fluorescence depolarization experiments on molecules embedded in a stretched anhydrous nitrocellulose matrix. The absorption transition moments lies almost parallel to the elongated axis of the molecule, but the emission transition moment makes an angle of 20° with the axis. The orientational distribution of the molecules in the polymer indicates significant deviations from a circular form.     

Denaturation of human serum albumin initiated by cetyltrimethylammonium bromide as monitored via the intrinsic fluorescence of the protein and fluorescence of an eosin probe

An analysis of the intrinsic fluorescence of the protein and the fluorescence of the eosin molecular probe in solutions of the protein was used to study the denaturation of human serum albumin (HSA) under the action of cetyltrimethylammonium bromide (CTAB), a cationic detergent, at various pH values. The denaturation of HSA under the influence of CTAB is a single-stage process over the entire pH range covered, pH (3.5–8.0). The maximum possible loosening of HSA globules in the presence of CTAB is reached at [CTAB] = 4 mmol/l. This detergent effectively denatures HSA at pH values higher than the pI of the protein (4.7).     

Fluorescent properties and spontaneous Raman spectroscopy of new ketocyanine probes in organic solvents

We have used fluorescence spectroscopy and spontaneous Raman spectroscopy to study the characteristics of two ketocyanine dyes: 2,5-di[(E)-1-(4-diethylaminophenyl)methylidene]-1-cyclopentanone (CPET) and 2-[(E)-1-(4-diethylaminophenyl)methylidene]-5-{(E)-1-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecalin) phenyl]methylidene}-1-cyclopentanone (CPMR) in organic solvents. The position of their electronic spectra depends strongly on the polarity of the solvent. We measured the dipole moments of the dyes in the equilibrium ground state and the Franck-Condon excited state. In mixtures of neutral nonpolar toluene with aprotic polar dimethylsulfoxide, we observe inhomogeneous broadening of the electronic spectra for the indicated compounds, due to fluctuations in solution of the intermolecular interaction energy. The time-resolved characteristics of fluorescence obtained suggest formation of an intermolecular hydrogen bond between the dye and the surrounding medium in a toluene-ethanol mixture. We measured the Raman spectra of CPET and CPMR in different organic solvents. The most intense lines in the 1582–1591 cm⁻¹ region can be assigned to stretching of the phenyl rings of the molecules; the lines in the 831–842 cm⁻¹ region can be assigned to a cyclopentanone ring mode; the lines at 1186–1195 cm⁻¹ can be assigned to stretching of the =C-C-bond of the phenyl ring and rocking of the H atoms of the phenyl ring. We have observed that the position and width of the lines for the stretching vibrations of the ketocyanines depend substantially on the polarity of the surrounding medium. The studied dyes can be used as probes for studying different biological systems by site-selective laser spectroscopy and Raman spectroscopy. The fact that these two methods can be used simultaneously for diagnostics of biosystems is an important advantage of ketocyanine dyes compared with other known probes. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 6, pp. 763–769, November–December, 2006.     

Luminescent spectral characteristics of eosin in solutions of human serum albumin when denatured by treatment with sodium dodecyl sulfate

From analysis of the fluorescence spectra of eosin molecules in a solution with human serum albumin (HSA), we have obtained information about the dynamics of protein conformational rearrangements during denaturing of the protein when treated with sodium dodecyl sulfate (SDS) for different pH values of the solution. We hypothesize that HSA denaturing in the presence of SDS occurs in two stages: the first stage is loosening of the protein globules, and the second stage is complete unfolding of the protein molecules. We have shown that denaturating of the protein in the presence of SDS passes through both stages for a solution pH below the isoelectric point of the albumin, while the denaturing stops in the first stage for a solution pH above the isoelectric point of the albumin. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 5, pp. 661–665, September–October, 2006.     

IR absorption anisotropy in perylene-3,4,9,10-tetracarboxylic acid dianhydride

Vacuum-deposited perylene-3,4,9,10-tetracarboxylic acid dianhydride thin films are investigated by IR-spectropolarimetry with variable incident angle of plane-polarized radiation. Anisotropy of absorption bands in the region 700–900 cm⁻¹ is detected. Absorption bands in the subregions 728–736 and 806–810 cm⁻¹ are shown to be of triplet nature with orthogonally oriented dipole moments within the triplet.     

Membrane Dipole Potential as Measured by Ratiometric 3-Hydroxyflavone Fluorescence Probes: Accounting for Hydration Effects

We previously applied the electrochromic modulation of excited-state intramolecular proton-transfer (ESIPT) reaction for the design of novel 3-hydroxyflavone (3-HF) derivatives as fluorescent probes for measuring the dipole potential, Ψ_D, in lipid bilayers (Klymchenko et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 11219). In the present work, this method was revisited to take into account the influence of the bilayer hydration on the emission ratiometric response of 3-HF probes. For this reason, it was necessary to deconvolute the whole fluorescence spectra into three bands corresponding to the non H-bonded forms, normal N^* and tautomer T^* forms, both participating to the ESIPT reaction, and to the H-bonded H–N^* form, excluded from this reaction. This allowed us to determine the pure N^*/T^* intensity ratio, without any contribution from the H–N^* form emission depending essentially on the bilayer hydration. This new approach allowed us to confirm the correlation we obtained between the response of 3-HF probes on dipole potential modifications and the corresponding response of the reference fluorescent probe di-8-ANEPPS, thus further confirming the potency of 3-HF probes as excellent emission ratiometric probes to measure dipole potential in lipid membranes.     

A combined theoretical and experimental study on molecular photonics

A detailed account of quantum chemical procedures for estimating the rate constants of the photophysical processes at work in polyatomic organic molecules is given. The results obtained from combined experimental and theoretical research into the spectral-luminescent properties of acridine, 9-aminoacridine, 2,7-dimethyl-9-diphenylaminoacridine, and of their protonated forms are reported. The electronic absorption and fluorescence spectra of acridine have been investigated at room temperature in ethanol solution of varying pH and in other solvents of different chemical nature and polarity. The energy of excited states, the rate constants of the deactivation of the excited states, and the dipole moments obtained by quantum chemical methods for the examined compounds are presented. The findings of the investigations are discussed. Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 10, pp. 93–107, October, 2008.