Jayanti Guharay

Protein–flavonol interaction: Fluorescence spectroscopic study

Recent studies have shown that various synthetic as well as therapeutically active naturally occurring flavonols possess novel luminescence properties that can potentially serve as highly sensitive monitors of their microenvironments in biologically relevant systems. We report a study on the interactions of bovine serum albumin (BSA) with the model flavonol 3‐hydroxyflavone (3HF), using the excited‐state proton‐transfer (ESPT) luminescence of 3HF as a probe. Upon addition of BSA to the flavonoid solutions, we observe remarkable changes in the absorption, ESPT fluorescence emission and excitation profiles as well as anisotropy (r) values. Complexation of 3HF with protein results in a pronounced shift (20 nm) of the ESPT emission maximum of the probe (from λ  maxem = 513 nm to λ  maxem = 533 nm) accompanied by a significant increase in fluorescence intensity. The spectral data also suggest that, in addition to ESPT, the protein environment induces proton abstraction from 3HF leading to formation of anionic species in the ground state. Fairly high values of anisotropy are observed in the presence of BSA for the tautomer (r = 0.25) as well as anion (r = 0.35) species of 3HF, implying that both the species are located in motion‐restricted environments of BSA molecules. Analysis of relevant spectroscopic data leads to the conclusions that two binding sites are involved in BSA–3HF interaction, and the interaction is slightly positively cooperative in nature with a similar binding constant of 1.1 − 1.3 × 105 M−1 for both these sites. Proteins 2001;43:75–81.

Excited-state intramolecular proton transfer (ESIPT) and charge transfer (CT) fluorescence probe for model membranes

Abstract Motivated by the burgeoning interest in designing and characterizing fluorescence probes with superior spectroscopic properties for exploring the structural and dynamic aspects of biological systems, we have investigated the photophysical behaviour of a synthetic flavonol, namely 4′- N , N -dimethylamino-3-hydroxyflavone (DMA3HF) in model membranes consisting of small unilamellar liposomes of the phospholipids DPPC and DMPC. The DMA3HF fluorophore is remarkable in the fact that it combines both excited-state proton transfer (PT) and charge transfer (CT) fluorescence emissions in a single molecular system. The nature and relevant parameters of the dual emission profiles, fluorescence excitation spectra, along with steady-state fluorescence anisotropy ( r ) and red edge excitation shift (REES) data have been used to probe the local environments of DMA3HF molecules in liposomes. Furthermore, r versus temperature ( T ) profiles are shown to provide estimates of thermotropic gel to liquid-crystalline state phase transition temperatures of the phospholipids, in excellent agreement with the existing literature data.

EXCITED STATE PROTON TRANSFER FLUORESCENCE OF 3-HYDROXYFLAVONE IN MODEL MEMBRANES

Abstract 3-Hydroxyflavone (3HF), the basic structural moiety of an important group of naturally occurring, biologically active flavonoids, has attracted extensive attention for its intramolecular excited-state proton-transfer (ESPT) and dual fluorescence characteristics. We report here, for the first time, its fluorescence (emission, excitation, polarization anisotropy and lifetime) behaviour in model membranes consisting of small, unilamellar liposomes of synthetic phosphatidylcholine (dimyristoyl phosphatidylcholine (DMPC) and dipalmitoyl phosphatidylcholine (DPPC)). The emission spectrum is conspicuously dominated by the ESPT tautomer fluorescence band. This result, along with other relevant data, indicates that the molecules are embedded in the lipid bilayer matrix facing environments where external H-bonding perturbation effects are minimized. The anisotropy ( r ) versus temperature ( T ) profiles dramatically reveal the thermotropic gel-liquid crystalline phase transition properties of the phospholipids, signifying a novel application of the intrinsic (tautomer) fluorescence of 3HF.

Influence of different environments on the excited-state proton transfer and dual fluorescence of fisetin

Abstract The influence of different protic and aprotic solvent environments on the excited-state intramolecular proton transfer (ESIPT) leading to a dual fluorescence behaviour of a biologically important, naturally occurring, polyhydroxyflavone, fisetin (3,3′,4′,7-tetrahydroxyflavone), has been investigated. The normal fluorescence band, in particular, is extremely sensitive to solvent polarity with ν max shifting from 24 510 cm −1 in dioxane ( E T (30)=36.0) to 20 790 cm −1 in methanol ( E T (30)=55.5). This is rationalized in terms of solvent dipolar relaxation process, which also accounts for the red edge excitation shifts (REES) observed in viscous environments such as glycerol at low temperatures. Significant solvent dependence of the tautomer fluorescence properties ( ν max, yield and decay kinetics) reveals the influence of external hydrogen bonding perturbation on the internal hydrogen bond of the molecule. These excited-state relaxation phenomena and their relevant parameters have been used to probe the microenvironment of fisetin in a membrane mimetic system, namely AOT reverse micelles in n -heptane at different water/surfactant molar ratio ( w 0 ).

Intramolecular excited-state proton transfer and charge transfer fluorescence of a 3-hydroxyflavone derivative in micellar media

Abstract The fluorescence emission behaviour of 4′- N,N -dimethylamino-3-hydroxyflavone (DMA3HF), has been examined in representative micellar media. The utility of this fluorophore as a novel micellar probe is demonstrated, exploiting various parameters of its excited-state intramolecular proton transfer (ESIPT) and charge transfer (CT) fluorescence. The intensity and polarization anisotropy of both ESIPT and CT fluorescence prove to be highly sensitive monitors for estimating the first and second critical micelle concentrations (CMC1 and CMC2). Furthermore, the CT fluorescence exhibits dramatic blue shifts in micelles, along with high values of polarization anisotropy and significant red edge excitation shift (REES). The results suggest that the fluorophore resides in highly polar, motionally constrained regions in the micelle interface.

Characterization of the fluorescence emission properties of prodan in different reverse micellar environments.

We have examined the steady state and time resolved fluorescence emission properties of the hydrophobic fluorescence probe, prodan, in three representative reverse micellar systems formed by the surfactants poly(oxyethylene) (tetramethylbutyl) phenylether (Triton X-100, neutral), cetyl trimethylammonium bromide (CTAB, cationic) and sodium bis-(2-ethylhexyl) sulfosuccinate (AOT, anionic) in organic solvent media containing different concentrations of water. The results obtained from the experiments indicate conspicuous dependence of the emission behaviour of prodan on the type of surfactant used and the water/surfactant molar ratio (w0). The nature of the emission profiles, along with relevant parameters namely emission maximum (lambda(em)max), anisotropy (r) and lifetime (tau) data are used to infer the distribution and microenvironments of the prodan molecules in the reverse micelles at different w0 values. Furthermore, quantitative estimates have been obtained for the polarities (in terms of the empirical polarity parameter E(T)(30)) of the sites of solubilization of the fluorophore in different reverse micellar systems.

Fluorescence polarization anisotropy as a novel tool for the determination of critical micellar concentrations

Abstract A new technique is proposed for the estimation of critical micellar concentrations (cmcs) based on fluorescence polarization anisotropy ( r ) measurements as a function of surfactant concentration ([surf]). Representative results are presented for Triton X 100 (TX 100), sodium dodecyl sulphate (SDS) and cetyl trimethyl ammonium bromide (CTAB). The first and second cmc values (cmc1 and cmc2) obtained from break points in r vs. [surf] plots are in reasonable agreement with existing literature data.

Excited-state proton-transfer and dual fluorescence of robinetin in different environments

Abstract Excited-state intramolecular proton-transfer (ESIPT) and dual emission behaviour of robinetin, (3,7,3′,4′,5′-pentahydroxy flavone) a naturally occurring flavonoid, have been examined in different protic and aprotic solvents and in aerosol-OT (AOT) reverse micelles. The normal fluorescence band, in particular, shows dramatic sensitivity to solvent polarity, with λmax shifting from 413 nm in dioxane (ET(30)=36.0) to 490 nm in methanol (ET(30)=55.5). This is interpreted in terms of a solvent dipolar relaxation mechanism, which also accounts for red edge excitation shifts (REES) noted in viscous environments, e.g. glycerol at low temperatures. The tautomer fluorescence properties (quantum yield and decay kinetics) also show significant solvent dependence. Relevant parameters of the dual emission bands have been used to infer the microenvironments of robinetin in AOT reverse micelles at different water/surfactant molar ratio (w0).

Luminescence behaviour of 5-hydroxyindole in different environments.

Steady state fluorescence emission spectroscopic studies along with some lifetime measurements have been performed for 5-hydroxyindole (5HI) in different environments. 5HI merits particular attention, since it is the chromophoric moiety of the non-natural amino acid 5-hydroxytryptophan (5HT), which has come into significant, recent prominence as a novel intrinsic optical probe for protein structure, function and dynamics. Studies in representative homogeneous solvents and solvent-mixtures indicate that unlike other fluorophores of related interest like indole (I) and 7-azaindole (7AI), the fluorescence emission maximum (lambda(em)max) of 5HI is relatively insensitive to solvent polarity. This behaviour suggests the lack of appreciable solvent dipolar relaxation in 5HI, which is consistent with our low temperature (77 K) emission data. Notwithstanding such limitation, fluorescence anisotropy (r) and quenching studies are shown to be effective for exploring changes in the micro-environments of 5HI in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT) reverse micellar assemblies (which serve as a biomembrane mimetic model system) with variation in water/surfactant molar ratio (w0).

An assessment of the usefulness of 5-hydroxytryptophan as an optical probe

Abstract 5-Hydroxytryptophan (5HT) has been the focus of much recent attention as a novel intrinsic fluorescence probe for proteins. Solvent-dependence studies reported here show that, compared to other fluorophores of related interest like tryptophan (Trp) and 7-azatryptophan (7AT), the fluorescence emission maximum (λemmax) of 5HT is relatively insensitive to solvent polarity. Thus, upon change of solvent from acetonitrile to water, the λemmax of 5HT fluorescence is red shifted by only 3 nm, in contrast to corresponding red shifts of much larger magnitudes observed for Trp and 7AT (14 and 23 nm, respectively). This behaviour suggests the lack of significant solvent dipolar relaxation effects in 5HT, which is corroborated by the low temperature emission measurements at 77°K. This aspect is also revealed in the fluorescence characteristics of 5HT in a membrane-mimetic model system, namely in AOT reverse-micellar assemblies containing different amounts of water. With increase in water content, the λemmax of 5HT remains almost unaffected, although fluorescence anisotropy and quenching data clearly indicate significant changes in the microenvironments of 5HT molecules.