Chemical and biological caging effects on the relaxation of a proton-transfer dye

We report studies of the interaction between a proton-transfer dye (1'-hydroxy,2'-acetonaphthone, HAN), with the human serum albumin (HSA) protein and a beta-cyclodextrin derivative (DM-beta-CD) in neutral water solutions. We used steady-state and picosecond time-resolved emission spectroscopy to follow the structural changes of HAN due to the hydrophobicity and confinement effect of these nanocavities. Upon encapsulation, the fluorescence intensity of the 1:1 inclusion complex in both cavities increases, and the emission lifetimes become longer. For the DM-beta-CD complexes, we obtained 430 and 920 ps, whereas for the HSA complexes we obtained 630 ps and 2 ns. Picosecond anisotropy measurements show strong confinement due to protein docking. The rotational time for the CD complex is 660 ps, whereas for the protein complex we find 6 ns. The process of energy transfer from the excited triptophan 214 (Trp214) of HSA to the trapped HAN occurs with high efficiency (71%), and the calculated distance between both chromophores is 17 A. We believe that the results are important for a better understanding of the processes occurring in inclusion complexes such as those in nanopharmacodynamics.     

Effect of cyclodextrin nanocavity confinement on the photorelaxation of the cardiotonic drug milrinone

We report on steady-state UV-visible absorption, emission, and picosecond emission studies of milrinone (MIR) drug in neutral water and complexed to cyclodextrins (alpha-, beta-, gamma-CD and dimethyl-beta-CD (DM-beta-CD)). The results reveal that MIR forms a 1:1 inclusion complex with CD. Upon encapsulation the emission intensity increases and the fluorescence lifetime changes from approximately 65 ps to 240-350 ps, indicating a confinement effect of the nanocages on the photophysical behavior of the drug. Due to its methyl groups, the DM-beta-CD complex shows the largest effect. The time-anisotropy experiments support the formation of 1:1 inclusion complexes and indicate motion of the drug inside the nanocavity. Furthermore, results of PM3 calculations combined with spectral and dynamical data show that the drug is not fully embedded into the cavities, and the conformation of the included complex explains the relatively short lifetimes and low emission quantum yields of these entities.     

Effect of nanocavity confinement on the relaxation of anesthetic analogues: relevance to encapsulated drug photochemistry

We report on UV-vis absorption and picosecond emission studies of methyl 2-amino-4,5-dimethoxy benzoate in neutral water and complexed to alpha-, beta-, and gamma-cyclodextrin (CD). Upon encapsulation, the emission intensity and the fluorescence lifetime increase, indicating a hydrophobic effect of the nanocages on the photophysical behavior of the guest. beta-CD confinement shows the largest effect. The time-dependent frequency shift of the emission (approximately 720 cm(-1)) in beta-CD nanocavity is larger than the one observed in water (approximately 490 cm(-1)) due to the hydrophobic and polarity effect of the nanocage and reflects a strengthening of the intramolecular H-bond of the encapsulated dye upon electronic excitation. Anisotropy measurements indicate a free motion of the guest into the nanocavity. The observed results are relevant to the hydrophobic as well as hydrophilic interactions which govern photochemistry and photophysics of caged drugs, organic, and biological systems.     

Role of xanthurenic acid 8-O-beta-D-glucoside,a novel fluorophore that accumulates in the brunescent human eye lens

We have been able to identify a blue fluorophore from the low-molecular weight soluble fraction of human adult nondiabetic brunescent cataract lenses as xanthurenic acid 8-O-beta-D-glucoside (XA8OG) (excitation = 338 nm and emission = 440 nm). To determine the role of this fluorophore in the lens, we have examined its photophysical and photodynamic properties. We found XA8OG to have a fluorescence quantum yield (phi) of 0.22 and a major emission lifetime of 12 ns. We found it to be a UVA-region sensitizer, capable of efficiently generating singlet oxygen species but little of superoxide. We also demonstrated that XA8OG oxidizes proteins when irradiated with UVA light, causing photodynamic covalent chemical damage to proteins. Its accumulation in the aging human lens (and the attendant decrease of its precursor O-beta-D-glucoside of 3-hydroxykynurenine) can, thus, add to the oxidative burden on the system. XA8OG, thus, appears to be an endogenous chromophore in the lens, which can act as a cataractogenic agent.     

Interaction of pyrene-end-capped poly(ethylene oxide) with bovine serum albumin and human serum albumin in aqueous buffer medium: a fluorometric study

The photophysical behavior of a hydrophobically tailored water-soluble polymer, pyrene-end-capped poly(ethylene oxide) (PYPY), has been studied in aqueous buffered bovine serum albumin (BSA) and human serum albumin (HSA) media. In buffered aqueous solution the polymer shows dual emission corresponding to the monomer and the excimer of pyrene moiety. The relative intensity of the monomer to the excimer emission shows interesting variation with the addition of BSA and HSA and is indicative of significant interaction of these albumin proteins with the polymer. The binding interaction has been shown to have a prominent role on the steady state fluorescence anisotropy of the two emission bands. Attempt has been made to determine the micropolarities of the protein microenvironments from a comparison of the variation of the monomer to excimer relative fluorescence intensities of the probe in water–dioxane mixtures with varying composition.     

Spectroscopic studies on the effect of temperature on pH-induced folded states of human serum albumin

Human serum albumin (HSA) is a very important multi-domain transporter protein in the circulatory system responsible for carriage of various kinds of ligands within the physiological system. HSA is also known to undergo conformational transformation at different pH(s) and temperatures. In this report we have studied the binding interactions of a photosensitizing drug, protoporphyrin IX (PPIX) with various conformers of HSA at different temperatures using picosecond time-resolved fluorescence spectroscopy. Also, using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy we have followed the structural transition of various conformers of HSA at different temperatures. Ensuring the intact binding of PPIX to various conformers of HSA at different temperatures as revealed through time-resolved fluorescence anisotropy decay and significant spectral overlap of emission of Trp214 residue (donor) in domain-IIA and absorption of PPIX (acceptor) bound to domain-IB of HSA, we have applied F?rster's resonance energy transfer (FRET) technique to determine the interdomain separation under various environmental conditions. The alkali-induced conformer of HSA shows almost no change in donor-acceptor distance in contrast to the native and acid-induced conformers of HSA, which show a decrease in distance with increase in temperature. Through this study the non-covalently bound PPIX is shown to be an efficient FRET probe in reporting the different temperature-induced folded states of HSA in buffer solutions of widely differing pH values.     

Cyclometalated platinum(II) complex with strong and broadband nonlinear optical response

A cyclometalated platinum(II) 4,6-diphenyl-2,2'-bipyridyl pentynyl complex (1) has been synthesized and structurally characterized. Its photophysical and third-order nonlinear optical properties have been systematically investigated. This complex exhibits a metal-to-ligand charge-transfer (1MLCT) absorption band between 400 and 500 nm and a 3MLCT emission band at approximately 591 nm at room temperature with a lifetime of approximately 100 ns. At 77 K, the emission band blue shifts. Both UV-vis absorption and emission spectra show solvent dependence. Low-polarity solvents cause a bathochromic shift of the absorption and emission bands. This complex also exhibits a broad and strong transient absorption from the near-UV to the near-IR spectral region, with a triplet absorption coefficient of 4933 L mol(-1) cm(-1) at 585 nm and a quantum yield of 0.51 for the formation of the triplet excited state. Nonlinear transmission and Z-scan techniques were employed to characterize the third-order nonlinearities of this complex. A strong and broadband reverse saturable absorption was observed for nanosecond and picosecond laser pulses due to the reduced ground-state absorption in the visible spectral range. It also exhibits a self-defocusing effect at 532 nm for nanosecond laser pulses. The excited-state absorption cross section deduced from the open-aperture Z-scan increases at longer wavelengths, with an exceptionally large ratio of excited-state absorption to ground-state absorption of 160 at 570 nm for picosecond laser pulses.     

Axial halogen ligand effect on photophysics and optical power limiting of some indium naphthalocyanines

Three axially substituted complexes, 2,3-octa(3,5-di-tert-butylphenoxy)-2,3-naphthalocyaninato indium chloride (1a), 2,3-octa(3,5-di-tert-butylphenoxy)-2,3-naphthalocyaninato indium bromide (1b), and 2,3-octa(3,5-di-tert-butylphenoxy)-2,3-naphthalocyaninato indium iodide (1c) have been synthesized and their photophysical properties have been investigated. Optical power limiting of nanosecond (ns) and picosecond (ps) laser pulses at 532 nm using these complexes has been demonstrated. All complexes display strong Q(0,0) absorption and measurable emission in the near-infrared region and exhibit strong excited-state absorption in the range of 470-700 nm upon ns laser excitation. The different axial ligands show negligible effect on the linear absorption, emission, and transient difference absorption spectra. However, the excited-state lifetime, triplet excited-state quantum yield, and efficiency to generate singlet oxygen are affected significantly by the heavier axial ligand. Brominated and iodinated complexes 1b and 1c show higher triplet excited-state quantum yield, while chlorinated complex 1a has longer excited-state lifetime and is more efficient in generating singlet oxygen. The iodinated complex 1c displayed the best optical limiting due to the higher ratio of excited-state absorption cross section to ground state absorption cross section (sigma(eff)/sigma(0)).     

Inclusion of Paracetamol into β-cyclodextrin nanocavities in solution and in the solid state

We report on steady-state UV-visible absorption and emission characteristics of Paracetamol, drug used as antipyretic agent, in water and within cyclodextrins (CDs): β-CD, 2-hydroxypropyl-β-CD (HP-β-CD) and 2,6-dimethyl-β-CD (Me-β-CD). The results reveal that Paracetamol forms a 1:1 inclusion complex with CD. Upon encapsulation, the emission intensity enhances, indicating a confinement effect of the nanocages on the photophysical behavior of the drug. Due to its methyl groups, the Me-β-CD shows the largest effect for the drug. The observed binding constant showing the following trend: Me-β-CD>HP-β-CD>β-CD. The less complexing effectiveness of HP-β-CD is due to the steric effect of the hydroxypropyl-substituents, which can hamper the inclusion of the guest molecules. The solid state inclusion complex was prepared by co-precipitation method and its characterization was investigated by Fourier transform infrared spectroscopy, 1H NMR and X-ray diffractometry. These approaches indicated that Paracetamol was able to form an inclusion complex with CDs, and the inclusion compounds exhibited different spectroscopic features and properties from Paracetamol.     

Effect of nanocavity confinement on the rotational relaxation dynamics: 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine in micelles

In continuation of our recent study on the steady state photophysics of a biologically active beta-carboline derivative, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), in the present article we have investigated the effect of nanocavity confinement on the excited state dynamics and rotational relaxation of the probe using picosecond time resolved fluorescence and fluorescence anisotropy techniques. The polarity dependent intramolecular charge transfer process is responsible for the remarkable sensitivity of this biological fluorophore in micellar environments. The fluorescence anisotropy decay of AODIQ incorporated inside the micelle is biexponential. The rotational motion of the probe was interpreted on the basis of a two step model consisting of a fast restricted rotation of the probe and a slow lateral diffusion of the probe in the micelle; both coupled to the overall rotation of the micelle. Experimental results reveal that micellar environment causes significant retardation of both the wobbling as well as the translational motion of the probe.     

Effect of denaturants on the stability of light-harvesting complex

 The effect of denaturants such as urea and normal alcohols on the formation of light-harvesting (LH) polypeptides/bacteriochlorophyll a (BChla) complex (LH1 complex) in n-octyl-β-D-glucopyranoside (OG) micelle was examined to provide an insight into stability of the complex. The stabilities of the LH1 complex in OG micelle and of the complex in the chromatophore of photosynthetic bacteria were compared by addition of denaturants. The extent of stability of these complexes was monitored by the change in absorbance of Qy band of BChla in these complexes, resulting generally in the blue-shifting of the Qy band from near 870 nm to about 777 nm upon addition of these denaturants. Urea and guanidium hydrochloride (Gnd) showed a relatively weak denaturing effect. Normal alcohols showed stronger denaturing effect, depending on the hydrophobicity of the alcohols. These results imply that the stability of LH1 complex in OG micelle can be largely attributed to the hydrophobic interactions in the complex as well as that of the complex in the chromatophore of photosynthetic bacteria. Received: 23 May 1997 Accepted: 13 September 1997     

Ultrafast photoinduced deligation and ligation dynamics: DCM in micelle and micelle-enzyme complex

We report studies on diffusion controlled deligation and ligation dynamics of a probe ligand 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl) 4H-pyran (DCM) with cationic cetyltrimethylammonium bromide (CTAB) micelles. In order to investigate the effect of spatial heterogeneity on the dynamics we study the DCM labeled micelle upon complexation with an enzyme alpha-chymotrypsin (CHT). The variation of fluorescence line-width (Gamma(t)) of DCM in the complex and also in the micelle indicates the diffusion dynamics of DCM through various environments of different polarities. The temporal behavior of Gamma(t) reveals that at 50 mM CTAB concentration the excited DCM traverses 6.5 Angstrom distance from the surface of a host micelle (deligation) before entering to a stern layer of another adjacent micelle (ligation). From neutron scattering experiment the distance 6.5 Angstrom is found to be the thickness of a stern layer of CTAB micelle. No indication of ligation has been found at 2 mM CTAB concentration as the intermicellar distance is estimated to be very large (416 Angstrom) compared to the previous case. The dynamical behavior of Gamma(t) is also indicative of significantly slower diffusion of the ligand molecules (DCM) at the surface of the micelle in presence and absence of the enzyme compared to that in the bulk buffer. We have also studied the dynamics of solvation and local geometrical restriction on the probe DCM at the micellar surface with and without CHT. With picosecond time resolution, we found time constants of the solvation relaxation processes of the DCM labeled enzyme-micelle complex to be 230 ps (45%) and 870 ps (55%), which were comparable to those of the micelle without the enzyme. The time dependent anisotropy revealing local orientational motions of the probe in the complex was also found to be similar to that of DCM at the micellar surface in absence of CHT. These studies attempt to link the dynamical features for insight into the ligand mediated intercellular communication and the biological function of the enzyme alpha-chymotrypsin upon complexation with the CTAB micelle.     

Inclusion complex of n-octyl beta-D-glucopyranoside and alpha-cyclodextrin in aqueous solutions: thermodynamic and structural characterization

Complex formation between octyl beta-D-glucopyranoside (OG) and alpha-cyclodextrin (alphaCD) was investigated on the basis of three highly accurate and appropriate experimental techniques. First, surface tension measurements showed that alphaCD directly acts on the surfactant monomers in the aqueous phase, leading to progressive depletion of the air-water interface with increasing cyclodextrin contents. Significant shift of OG critical micelle concentration (cmc) was consequently observed: the higher alphaCD concentration, the higher the cmc value. Experiments performed at surfactant and cyclodextrin concentrations in the Gibbs regime of surface tension versus OG content were performed on one hand to establish Job's plot that showed 1:1 stoichiometry of the OG-alphaCD complex and on the other hand to calculate the association constant found equal to (1.85 +/- 0.35) x 10(3) L mol(-1). An inclusion process of the surfactant alkyl residue within the cyclodextrin cavity was confirmed by one-dimensional (1)H NMR, and the structure of the mixed assembly was extensively characterized by two-dimensional NOESY (1)H NMR. OG penetrates alphaCD so that its hydrocarbon chain is embedded inside the cyclodextrin cavity, and its polar head as well as the alpha-methylene group emerges outside the alphaCD secondary face. Solubility behavior of the OG-alphaCD complex in a wide range of host-guest ratios and concentrations was finally examined by turbidity recording and optical microscopy. At very low free cyclodextrin levels in the solution, the complex presented high solubility behavior up to more than 70 mM. By increasing nonassociated alphaCD in the mixture, propensity of the cyclodextrin molecules to crystallize was observed at concentrations far below the 100 mM aqueous solubility of the pure cyclodextrin. The hexagonal shape of the crystals seen in the optical microscopy images suggested they were, partially at least, composed of the solid complex.     

Fluorimetric study on the charge transfer behavior of trans-ethyl-(p-dimethylamino cinnamate) and its derivative in cyclodextrin cavities

The intramolecular charge transfer (ICT) property of trans-ethyl p-(dimethylamino) cinnamate (EDAC) and its acid derivative, p-(dimethylamino) cinnamic acid (DMACA), is used to monitor the encapsulation behavior of these probes into the cyclodextrin (CD) nanocavities by steady state and picosecond time-resolved fluorescence spectroscopy. The ICT fluorescence band intensity was found to increase with concomitant blue shift in presence of cyclodextrins. The encapsulation behavior was further characterized by increase in emission yield, fluorescence anisotropy as well as lifetime values. Detailed analysis of the spectroscopic data indicate that the probes enter through the dimethyl amino group pointing to the secondary rim of the doughnut-shaped hydrophobic cavities to form 1:1 complex at different pH, however, the extent of penetration is more for EDAC compared with DMACA.     

Ultrafast surface solvation dynamics and functionality of an enzyme alpha-chymotrypsin upon interfacial binding to a cationic micelle

In this contribution we report studies on enzymatic activity of alpha-chymotrypsin (CHT) upon complexation with cationic cetyltrimethylammonium bromide (CTAB) micelle. With picosecond time resolution, we examined solvation dynamics at the interface of CHT-micelle complex, and rigidity of the binding. We have used 5-(dimethyl amino) naphthalene-1-sulfonyl chloride (dansyl chloride; DC) that is covalently attached to the enzyme at the surface sites. The solvation processes at the surface of CHT in buffer solution are found to be mostly in the sub-50 ps time scale. However, at the interface the solvation correlation function decays with time constant 150 ps (65%) and 500 ps (35%), which is significantly different from those found at the enzyme and micellar surfaces. The binding structure of the enzyme-micelle complex was examined by local orientational motion of the probe DC and compared with the case without micelle. The orientational dynamics of the probe DC in the complex reveals a structural perturbation at the surface sites of CHT upon complexation, consistent with other reported structural studies. We also found possible entanglement of charge transfer dynamics of the probe DC on the measured solvation processes by using time-resolved area normalized emission spectroscopic technique. The interfacial solvation process and complex rigidity elucidate the strong recognition mechanism between CHT and the micelle, which is important to understand the biological function of CHT upon complexation with the micelle.     

Molecular dynamics simulations of GlpF in a micelle vs in a bilayer: conformational dynamics of a membrane protein as a function of environment

Octyl glucoside (OG) is a detergent widely employed in structural and functional studies of membrane proteins. To better understand the nature of protein-OG interactions, molecular dynamics simulations (duration 10 ns) have been used to explore an alpha-helical membrane protein, GlpF, in OG micelles and in DMPC bilayers. Greater conformational drift of the extramembraneous protein loops, from the initial X-ray structure, is seen for the GlpF-OG simulations than for the GlpF-DMPC simulation. The mobility of the transmembrane alpha-helices is approximately 1.3x higher in the GlpF-OG than the GlpF-DMPC simulations. The detergent is seen to form an irregular torus around the protein. The presence of the protein leads to a small perturbation in the behavior of the alkyl chains in the OG micelle, namely an approximately 15% increase in the trans-gauche(-)-gauche(+) transition time. Aromatic side chains (Trp, Tyr) and basic side chains (Arg, Lys) play an important role in both protein-detergent (OG) and protein-lipid (DMPC) interactions.     

Relaxometric and modelling studies of the binding of a lipophilic Gd-AAZTA complex to fatted and defatted human serum albumin

A new lipophilic gadolinium chelate consisting of a long aliphatic chain bound to the AAZTA coordination cage (Gd-AAZTAC17) has been synthesised. It possesses two coordinated water molecules (q=2) in fast exchange with the solvent (tau298(M) = 67 ns), which yields a relaxivity of 10.2 mM(-1) s(-1). At concentrations greater than 0.1 mM, it forms micelles (average diameter 5.5 nm) characterised by a relaxivity of approximately 30 mM(-1) s(-1) at 20 MHz and 298 K. The latter value appears to be "quenched" by magnetic interactions among the Gd(III) ions on the surface of the micelle that cause a decrease in the electronic relaxation time. A relaxivity of 41 mM(-1) s(-1) was recorded for this micellar system when 98 % of the Gd(III) ions were replaced by diamagnetic Y(III). Gd-AAZTAC17 exhibits a better affinity for fatted human serum albumin (HSA) than for defatted HSA, whereas the relaxivities of the supramolecular adducts are reversed. The relaxivity shown by Gd-AAZTAC17/defatted HSA ({r b(1) (20 MHz, 298 K)=84 mM(-1) s(-1)) is by far the highest relaxivity reported so far for non-covalent paramagnetic adducts with slow-moving substrates. As shown by molecular docking calculations, the gadolinium complex enters a hydrophobic pocket present in fatted HSA more extensively than the corresponding adduct with defatted HSA. Interestingly, no marked difference was observed in either the relaxation enhancement or the binding affinity between fatted and defatted HSA when the binding titrations were carried out at a Gd-AAZTAC17 concentration higher than its critical micellar concentration (cmc). This behaviour has been attributed to the formation of an association between the negatively charged micelle of the lipophilic metal complexes and the positive residues on the surface of the protein.     

Fluorescence of 1,3-Di(1-pyrenyl)propane probe incorporated into human serum albumin protein enforced conformations of the probe

 Steady-state and time-resolved fluorescence spectra of 1,3-di(1-pyrenyl)propane (1Py-(3)-1Py) incorporated into macromolecules of human serum albumin (HSA), into micelles of dodecyltrimethylammo-nium chloride (DTAC), and dissolved in 1,4-dioxane were compared. The steady-state fluorescence spectra indicated that in all the mentioned environments, upon excitation of 1Py-(3)-1Py, light was emitted from the single pyrene chromophores (1Py^*) and from the 1Py, 1Py^* excimers. The time-resolved fluo-rescence emission registered at 480 nm (excimer emission) for 1Py-(3)-1Py in the DTAC micelles and dissolved in 1,4-dioxane allowed to monitor formation of excimer with time constant τ₁=40.0 ns and 9.6 ns, for 1Py-(3)-1Py in the DTAC micelles and in 1,4-dioxane, respectively. However, when the 1Py-(3)-1Py probe was located inside of the macromolecules of HSA, only the decay of emission was observed for excimer with our set-up (t>2 ns after excitation). The instantaneous formation of excimer, unrelated to the decay of monomer excitation, indicates that the considerable fraction of 1Py-(3)-1Py in the hydrophobic pockets of HSA is present as the ground state dimer. The red shift (Δλ=8 nm) and broadening of UV absorption for 1Py-(3)-1Py in HSA (when compared with absorption 1Py-(3)-1Py in 1,4-dioxane) and comparison of exci-tation spectra of 1Py-(3)-1Py in HSA and in 1,4-dioxane also indicate that label molecules bound to some sites of HSA are in the ground state in the dimer conformation. Moreover, the close values of the ratios of intensities of monomer emission to excimer emission, registered 2 ns (5 ns gate) after excitation pulse with duration 300 ps and at the steady-state conditions, indicate that the interconversion between conformers of 1Py-(3)-1Py inside of the macro-molecules of HSA is slow in comparison with the decay time of Py chromophore in the excited state in HSA (two-exponential decay with decay times τ₁=2.41 ns, τ₂=69.0 ns). Thus, ratios of the intensities of monomer and excimer emissions of 1Py-(3)-1Py in HSA do not allow to obtain any information on the local microfluidity inside of the protein macromolecules but could be used for discrimination between different conformations of the probe, possibly located in different protein pockets. Received: 29 April 1996 Accepted: 15 August 1996     

Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes

This study is aimed at establishing optimal conditions for the use of 2,2'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (tau approximately 2.5 ns and = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 0.05-0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.     

The role of tautomeric and rotameric species in the photophysics of 2-(2′-hydroxyphenyl)benzoxazole

The complex photophysical behaviour of 2-(2′-hydroxyphenyl)benzoxazole (HBO) has been investigated using both steady-state absorption and fluorescence and time-resolved (picosecond) emission spectroscopy, and found to be consistent with the existence of various tautomeric and rotameric species in equilibrium in the ground state. The natures of these species are discussed. Molecular-orbital calculations (PPP), performed for all species, gave results in good agreement with experiment. Excited-state intramolecular proton transfer was found to occur with a rate constant exceeding 1 × 10¹¹ s^?1.