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 glycation on the structure and dynamics of DNA: a critical spectroscopic approach.

Glycated DNA is considered to be a pathogenic factor for diabetes mellitus. Here we present a novel and preliminary study on normal and glycated (with fructose and glucose-6-phosphate as reducing sugars) human placenta DNA using agarose gel electrophoresis and photon correlation spectroscopy. The former is used to find structural alterations, while the latter is exploited to observe differences in the dynamics between normal (i.e., pure) and glycated DNA molecules. For scattering angles up to 90 degrees , we obtained a quasi-single-exponential relaxation process for the pure DNA, whereas at higher scattering angles the relaxation of pure DNA becomes broader with a stretching parameter beta approximately 0.6 at 130 degrees. Interestingly, for both the glycated DNAs stretched relaxation profiles and higher relaxation rates (Omega) are observed for all scattering angles. Moreover, a separate and very fast relaxation (e.g., relaxation time tau approximately 2 micros at 90 degrees ) can be noticed for both the glycated DNAs at all the studied scattering angles. Thus, the dramatic changes in the relaxation parameters (Omega, tau, and beta) of the glycated DNA show at the molecular level, for the first time, that the structure and dynamics of DNA are strongly affected by glycation. Implications of the results are discussed.

Interaction of flavonoids with red blood cell membrane lipids and proteins: antioxidant and antihemolytic effects.

Plant flavonoids are emerging as potent therapeutic drugs effective against a wide range of free radical mediated diseases. Hence their interactions with cell membranes, which generally serve as targets for lipid peroxidation, are of enormous interest. Here we report in vitro studies, via absorption and fluorescence spectroscopy, on the effects of several flavonoids (namely fisetin, quercetin, chrysin, morin, and 3-hydroxyflavone, 3-HF) in goat RBC membranes. Owing to the presence of functionally relevant membrane protein components embedded in the lipid bilayer RBC ghosts provide a more realistic system for exploring drug actions in biomembranes than simpler membrane models like phosphatidylcholine liposomes used in our previous studies [e.g. B. Sengupta, A. Banerjee, P.K. Sengupta, FEBS Lett. 570 (2004) 77-81]. Here, we demonstrate that binding of the flavonoids to the RBC membranes significantly inhibits lipid peroxidation, and at the same time enhances their integrity against hypotonic lysis. Interestingly, the antioxidant and antihemolytic activities are found to be crucially dependent on the locations of the flavonoids in the membrane matrix as revealed by fluorescence studies. Furthermore, we observe that FRET (from membrane protein tryptophans to flavonoids) occurs with significant efficiency indicating that the flavonoid binding sites lie in close proximity to the tryptophan residues in the ghost membrane proteins.

Exploring the antioxidant property of bioflavonoid quercetin in preventing DNA glycation: a calorimetric and spectroscopic study.

Reducing sugars for example glucose, fructose, etc., and their phosphate derivatives non-enzymatically glycate biological macromolecules (e.g., proteins, DNA and lipids) and is related to the production of free radicals. Here we present a novel study, using differential scanning calorimetry (DSC) along with UV/Vis absorption and photon correlation spectroscopy (PCS), on normal and glycated human placenta DNA and have explored the antioxidant property of the naturally occurring polyhydroxy flavone quercetin (3,3',4',5,7-pentahydroxyflavone) in preventing the glycation. The decrease in the absorption intensity of DNA in presence of sugars clearly indicates the existence of sugar molecules between the two bases of a base pair in the duplex DNA molecule. Variations were perceptible in the PCS relaxation profiles of normal and glycated DNA. The melting temperature of placenta DNA was decreased when glycated suggesting a decrease in the structural stability of the double-stranded glycated DNA. Our DSC and PCS data showed, for the first time, that the dramatic changes in the structural properties of glycated DNA can be prevented to a significant extent by adding quercetin. This study provides valuable insights regarding the structure, function, and dynamics of normal and glycated DNA molecules, underlying the manifestation of free radical mediated diseases, and their prevention using therapeutically active naturally occurring flavonoid quercetin.

Properties of normal and glycated human hemoglobin in presence and absence of antioxidant.

We present a novel approach to study properties of normal (HbA) and nonenzymatically glycated (HbA(Ic), HbA(Ia+b)) human hemoglobin using absorption spectroscopy and differential scanning calorimetry. The effect of the presence of the antioxidant fisetin on glycation of HbA is studied. Here, absorption spectroscopy has been fruitfully exploited to observe the formation of the glycated hemoglobin. With the differential scanning calorimetry, we studied the thermal unfolding of the protein hemoglobin at various conditions. The thermogram of the pure HbA showed two transition regions, with the occurrence of a partially unfolded intermediate state (the formation of which is mainly reversible) prior to complete denaturation (irreversible process). The denaturation temperature of HbA was found to be strongly dependent on the heating rate. Furthermore, there is a significant cooperativity between the two transition regions in pure HbA. The overall denaturation for the glycated hemoglobin takes place at a lower temperature, suggesting a decrease in the stability of the protein when it is glycated. In presence of fisetin, glycation is inhibited to a certain extent and the thermograms match well with that of normal HbA. Implications of the results are discussed.

Interactions of the plant flavonoid fisetin with macromolecular targets: insights from fluorescence spectroscopic studies.

Fisetin (3,7,3',4'-tetrahydroxyflavone) is a bioactive plant flavonoid of immense importance as a potentially useful therapeutic drug, for various free radical mediated as well as other diseases. In a recent paper, we demonstrated the novel uses of the exquisitely sensitive intrinsic fluorescence of this compound to explore its binding characteristics in liposomal membranes [B. Sengupta, A. Banerjee, P.K. Sengupta, Investigations on the binding and antioxidant properties of the plant flavonoid fisetin in model biomembranes, FEBS Lett. 570 (2004) 77-81]. Here, we have exploited this technique to examine its interactions with relevant macromolecular targets, namely double stranded DNA (from calf thymus), and the physiologically important circulatory protein, Human Serum Albumin (HSA). In the presence of DNA dramatic changes are observed in the intrinsic fluorescence behaviour of fisetin. These, along with other relevant supporting spectroscopic data, suggest that fisetin binds intercalatively between the base pairs of DNA. From the studies on fisetin-HSA interaction, the existence of two distinct binding sites are inferred. Furthermore we present evidence for the occurrence of efficient Förster type fluorescence resonance energy transfer from tryptophan to fisetin, indicating that both binding sites of fisetin in HSA are proximal to the unique tryptophan - 214 residue present in the interdomain (between IIA and IIIA domains) loop region of the protein.

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.

Characterization of serotonin in protein and membrane mimetic environments: a spectroscopic study.

As a first step toward using the photophysical properties of serotonin to probe its interactions with biological target sites, we have examined its interactions with human serum albumin (HSA), chosen as a surrogate for the actual receptor proteins in physiological systems, and with sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/heptane/water reverse micelles, chosen as a biomembrane mimetic environment for the transmembrane portion of the receptor protein. Although the emission maximum of serotonin is relatively insensitive to the polarity of the local environment, which is attributed to lack of solvent dipolar reorientation of the 5-hydroxyindole chromophore, the fluorescence anisotropy (r) served as a useful and sensitive parameter from which the binding constants (K) and Gibbs energy changes (deltaG) were estimated for serotonin-HSA and serotonin-AOT reverse micellar interactions. Fluorescence-decay studies of serotonin show double-exponential kinetics in homogeneous aqueous solvent due to the structural heterogeneity arising from different rotamers of serotonin. In contrast, upon binding to HSA, a single-exponential fluorescence-decay profile was observed indicating the occurrence of a single structural species of serotonin in the protein environment. Furthermore, far-UV-circular-dichroism (CD) spectroscopic data indicate that the secondary structural features of HSA remain essentially intact after binding to serotonin. This preliminary research can be expected to open the door to extensive future studies on interactions of serotonin with relevant target proteins and associated cell membranes involved in its diverse physiological functions.

Binding of quercetin with human serum albumin: a critical spectroscopic study.

Flavonols are plant pigments that are ubiquitous in nature. Quercetin (3,3',4',5,7-pentahydroxyflavone) and other related plant flavonols have come into recent prominence because of their usefulness as anticancer, antitumor, anti-AIDS, and other important therapeutic activities of significant potency and low systemic toxicity. Quercetin is intrinsically weakly fluorescent in aqueous solution, showing an emission maximum at approximately 538 nm. Upon binding to human serum albumin (HSA), quercetin undergoes dramatic enhancement in its fluorescence emission intensity, along with the appearance of dual emission behavior, consisting of normal and excited-state proton transfer (ESPT) fluorescence. In addition, the occurrence of a third emitting species has been noted for the first time. This is attributed to a electronic ground-state complex formed in the protein environment. High values of the fluorescence anisotropy (r) are obtained in the presence of HSA for the ESPT tautomer (r = 0.18), as well as the complex species (r = 0.37) of quercetin, indicating that the precursor ground-state molecules for both these emitting species of quercetin molecules are located in the motionally constrained sites of HSA. The steady-state emission data suggest that quercetin binds to two distinct sites in HSA from which the emissions from the normal tautomer and complex species take place. The preliminary results of studies on emission decay kinetics are also reported herein. Studies by far-UV circular dichroism spectroscopy reveal that binding of quercetin induces no significant perturbation in the secondary structure of HSA.

The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study.

Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient Förster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.

Low temperature luminescence behaviours of 7-azatryptophan, 5-hydroxytryptophan and their chromophoric moieties.

The non-natural amino acids 7-azatryptophan (7AT) and 5-hydroxytryptophan (5HT) have come into significant recent prominence as novel intrinsic luminescence probes for protein structure, function and dynamics. Here, we examine the low temperature luminescence behaviours of these molecules and their respective chromophoric moieties 7-azaindole (7AI) and 5-hydroxyindole (5HI) in representative solvent media. To ascertain, in particular, the potential usefulness of 7AT and 5HT as phosphorescence probes for exploring protein environments with different hydrogen bonding characteristics, a comparison is made of the phosphorescence properties of 7AI and 5HI chromophores in frozen solutions of ethanol and ethyl acetate at 77 K. These solvent media have been chosen as representative models for polar protic and aprotic environments in proteins, respectively. Our findings indicate that one or more of the phosphorescence emission parameters (phosphorescence emission maxima, relative yield and phosphorescence lifetime) of 7AI and 5HI chromophores can serve as sensitive and discriminating probes of hydrogen bonding and related aspects of their surrounding environments. Furthermore, in a model viscous environment (glycerol at low temperatures) significant temperature dependence and red edge excitation shift (REES) effects are observed for the fluorescence emission of 7AT and its chromophoric moiety 7AI. This is consistent with pronounced dipolar relaxation properties of these molecules, and suggests interesting possibilities for exploiting REES in exploring their environmental rigidity in motionally constrained situations.