Pradeep K. Sengupta

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.

Investigations on the binding and antioxidant properties of the plant flavonoid fisetin in model biomembranes

Plant flavonoids are emerging as potent therapeutic drugs for free radical mediated diseases, for which cell membranes generally serve as targets for lipid peroxidation and related deleterious effects. Screening and characterization of these ubiquitous, therapeutically potent polyphenolic compounds, require a clear understanding regarding their incorporation and possible location in membranes, as well as quantitative estimates of their antioxidative and radical scavenging capacities. Here, we demonstrate the novel use of the intrinsic fluorescence characteristics of the plant flavonoid fisetin (3,3′,4′,7‐OH flavone) to explore its binding and site(s) of solubilisation in egg lecithin liposomal membranes. Spectrophotometric assays have been used to obtain quantitative estimates of its antioxidative capacity. Furthermore, our quantum mechanical semi‐empirical calculations provide a quantitative measure for the free radical scavenging activity of fisetin from the OH (at 3, 3′, 4′, 7 positions of the molecule)‐bond dissociation enthalpies. Implications of these findings are discussed.

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.

Influence of different micellar environments on the excited-state proton-transfer luminescence of 3-hydroxyflavone

Abstract Excited-state proton-transfer and dual emission behaviour of 3-hydroxyflavone (3HF) have been investigated in representative micellar media. The relative yield of the green tautomer emission of 3HF shows pronounced enhancement in micelles, which suggests that the 3HF molecules are largely distributed in regions of low polarity, where external hydrogen-bonding perturbations are minimized. Further, the usefulness of the tautomer emission yield as a sensitive probe for estimation of critical micelle concentration values, is demonstrated. Preliminary results of studies on emission decay kinetics are also reported.

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.

Ground- and excited-state proton transfer and antioxidant activity of 3-hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies

Excited-state intramolecular proton transfer (ESIPT) and dual luminescence behaviour of 3-hydroxyflavone (3-HF) have been utilized to monitor its binding to liposomal membranes prepared from egg yolk phosphatydilcholine (EYPC). Additionally, absorption spectrophotometric assay has been performed to evaluate the antioxidant activity of 3-HF against lipid peroxidation in this membrane system. When 3-HF molecules are partitioned into EYPC liposomes, a weak long-wavelength absorption band with lambda(abs)(max) approximately 410 nm appears in addition to the principal absorption at approximately lambda(abs)(max) = 345 nm. Selective excitation of the 410 nm band produces the characteristic emission (lambda(em)(max) approximately 460 nm) of the ground-state anionic species, whereas excitation at the higher energy absorption band leads to dual emission with predominatly ESIPT tautomer fluorescence (lambda(em)(max) = 528 nm). Both ESIPT tautomer and the anionic species exhibit fairly high fluorescence anisotropy (r) values (r = 0.122 and 0.180, respectively). Biexponential fluorescence decay kinetics are observed for the ESIPT tautomer as well as the ground-state anionic forms, indicating heterogeneity in the microenvironments of the corresponding emitting species. Furthermore, we demonstrate that lipid peroxidation of EYPC liposomes is significantly inhibited upon 3-HF binding, suggesting that 3-HF can be potentially useful as an inhibitor of peroxidative damage of cell membranes.

Effect of reverse micelles on the intramolecular excited state proton transfer (ESPT) and dual luminescence behaviour of 3-hydroxyflavone

Abstract The excited-state proton transfer and dual emission behaviour of 3-hydroxyflavone (3HF) have been investigated in reverse micelles of sodium bis(2-ethylhexyl) sulphosuccinate (AOT)/ n -heptane at different values of water to surfactant molar ratio ( W O ). The green tautomet emission (λ max ≈ 524 nm) and blue-violet normal emission (λ max ≈ 400 nm) originate from two different ground state populations of 3HF molecules, which are located respectively in the apolar phase and at the interphase of the reverse micelles, proximal to the AOT head groups. With increasing W 0 the relative yield of the green emission band is enhanced with a concomitant decrease in that of the blue-violet emission. This is interpreted in terms of the population of 3HF molecules which are initially located in the interfacial region proximal to the polar head groups being “pushed” out into the apolar phase, where external hydrogen bonding perturbations are minimized.