Ground and excited state proton transfer and antioxidant activity of 7-hydroxyflavone in model membranes: absorption and fluorescence spectroscopic studies

Steady state and time resolved fluorescence spectroscopy have been used to probe microenvironments of the therapeutically active intrinsically fluorescent flavonoid, 7-hydroxyflavone (7-HF), in model membranes consisting of multilamellar phosphatidylcholine liposomes. Additionally, the antioxidant effects of 7-HF against lipid peroxidation have been evaluated using spectrophotometric assay. Large Stokes shifted emissions with distinct spectroscopic signatures, are observed from the excited state proton transfer (ESPT) tautomer (which is generated by a solvent mediated mechanism) and the ground state anion of 7-HF. The neutral (7-HFN) and anionic (7-HFA) species' appear to be located in the non-polar acyl chain and the polar head group regions of the lipid vesicles respectively. The partition coefficients of 7-HFN and 7-HFA in these vesicles have also been estimated using their intrinsic fluorescence. Anisotropy (r) versus temperature (T) measurements reveal the utility of the tautomer fluorescence anisotropy as a sensitive parameter for exploring structural changes in the membranes. Fluorescence decay kinetics studies indicate heterogeneity in the microenvironments of both 7-HFN and 7-HFA. Furthermore, we demonstrate that lipid peroxidation of the model membranes is partially arrested upon 7-HF binding, suggesting its potential usefulness as an inhibitor of peroxidative damage of cell membranes.     

A ratiometric fluorescent probe for alkaline phosphatase via regulation of excited‐state intramolecular proton transfer

A ratiometric fluorescent probe 2‐(benzimidazol‐2‐yl)phenyl phosphoric acid (1) for alkaline phosphatase (ALP) is designed and synthesized. The method employs the modulation of the excited‐state intramolecular proton transfer (ESIPT) process of 2‐(2'‐hydroxyphenyl)benzimidazole (HPBI) through the hydroxyl group protection/deprotection reaction. Upon phosphorylated with POCl₃, HPBI shows only an emission peak at 363 nm due to the blockage of ESIPT. However, once selective enzymatic hydrolysis with alkaline phosphatase (ALP) in Tris–HCl buffer occurs, the probe 1 is returned to HPBI and the ESIPT process is switched on, which results in a decrease in the emission band at 363 nm and an increase in a new fluorescence peak around 430 nm. The fluorescence intensity ratio at 430 and 360 nm (I₄₃₀/I₃₆₀) increases linearly with the activity of ALP up to 0.050 U/mL and the detection limit is 0.0013 U/mL. The proposed probe shows excellent specificity toward ALP. Copyright © 2015 John Wiley & Sons, Ltd.     

Excited state intramolecular proton transfer in 3-hydroxychromone: a DFT-based computational study

Potential energy (PE) curves for intramolecular proton transfer in the ground (GSIPT) and intramolecular proton transfer in the excited (ESIPT) states of 3-hydroxychromone (3HC) have been studied using DFT-B3LYP/6-31G(d,p) and TD-DFT/6-31G(d,p) level of theory, respectively. Our calculations suggest the non-viability of GSIPT in 3HC. Excited states PE calculations show the existence of ESIPT process in 3HC. ESIPT in 3HC has also been explained in terms of HOMO and LUMO electron densities of the enol and keto tautomers of 3HC.     

Effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption

This study examined the effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption. At the beginning of experiment (week 0), according to both the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, rats (n = 24) were divided into 3 groups and fed with a standard diet (group C), a diet containing ethanol (group E), or a diet containing ethanol and β‐carotene (group E+B). After 10 weeks, plasma AST and ALT, fat accumulation in the liver, antioxidant enzyme activities in erythrocytes and the liver, malondialdehyde (MDA), and α‐tocopherol and retinol in plasma and hepatic samples were analyzed. The chronic alcohol diet significantly increased AST and ALT levels in plasma, and these changes were prevented by supplementing the diet with β‐carotene. Glutathione (GSH) in erythrocytes and in the liver was significantly elevated in rats fed with a diet containing β‐carotene. The results indicate that β‐carotene supplementation can prevent ethanol‐induced liver damage and increase GSH concentrations in erythrocytes and the liver. Copyright © 2009 John Wiley & Sons, Ltd.     

Resonant and non‐resonant energy transfer from Ce3+ → X (X = Tb3+, Eu3+ or Dy3+) in NaMgSO4F material

An inorganic NaMgSO₄F fluoride material was prepared by the wet chemical method and studied for its photoluminescence (PL) and resonant–non‐resonant energy transfer (RET and NORET) capabilities between Ce³⁺ → Tb³⁺, Ce³⁺ → Eu³⁺ and Ce³⁺ → Dy³⁺ rare earth ions. The Tb³⁺ emission for Ce³⁺ → Tb³⁺ transfers under ultraviolet (UV) wavelengths peaked at 491, 547, and 586 nm, for excitation at 308 nm due to ⁵D₄ → ⁷F_J (J = 4, 5, 6) transitions. Eu emission spectra were observed at 440 nm (Eu²⁺), 593 nm and 616 nm (Eu³⁺) recorded for different concentrations of materials, whereas Dy³⁺ emission from Ce³⁺ → Dy³⁺ transfer under UV wavelengths peaked at 485 nm and 577 nm due to ⁴F_9/2 → ⁶H_15/2 and ⁶H_13/2 transitions. The purpose of the present study is to understand the RET and NORET effects of Tb³⁺, Eu³⁺ and Dy³⁺ co‐doping in a NaMgSO₄F:Ce³⁺ luminescent material, which could be used as a green‐emitting material for lamp phosphors.     

Biomolecular interaction study of hydralazine with bovine serum albumin and effect of β‐cyclodextrin on binding by fluorescence, 3D,synchronous, CD,and Raman spectroscopic methods

Spectrofluoremetric technique was employed to study the binding behavior of hydralazine with bovine serum albumin (BSA) at different temperatures. Binding study of bovine serum albumin with hydralazine has been studied by ultraviolet–visible spectroscopy, fluorescence spectroscopy and confirmed by three‐dimensional, synchronous, circular dichroism, and Raman spectroscopic methods. Effect of β‐cyclodextrin on binding was studied. The experimental results showed a static quenching mechanism in the interaction of hydralazine with bovine serum albumin. The binding constant and the number of binding sites are calculated according to Stern–Volmer equation. The thermodynamic parameters ?H^o, ?G^o, ?S^o at different temperatures were calculated. These indicated that the hydrogen bonding and weak van der Waals forces played an important role in the interaction. Based on the Förster's theory of non‐radiation energy transfer, the binding average distance, r, between the donor (BSA) and acceptor (hydralazine) was evaluated and found to be 3.95 nm. Spectral results showed that the binding of hydralazine to BSA induced conformational changes in BSA. The effect of common ions on the binding of hydralazine to BSA was also examined. Copyright © 2016 John Wiley & Sons, Ltd.     

Enzyme‐triggered fluorescence turn‐off/turn‐on of carbon dots for monitoring β‐glucosidase and its inhibitor in living cells

Energy transfer engineering based on fluorescent probes for directly sensing enzyme activities are in great demand as enzyme‐mediated transformations, which are central to all biological processes. Here, a fluorescence carbon dot (CD)‐based assay exhibiting selective responses to the quantitation of β‐glucosidase and the effect of its inhibitor was developed. The most common substrate, para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPG) was hydrolyzed by β‐glucosidase to release p‐nitrophenol (pNP), which can efficiently quench fluorescence of CDs via an inner filter effect and electron transfer. However, in the presence of inhibitors of β‐glucosidase, the fluorescence intensity gradually recovered as the concentration of inhibitors increased. Therefore, the enzyme‐triggered fluorescence turn‐off/turn‐on of specific CDs successfully achieved sensitive detection of β‐glucosidase and monitored the effect of its inhibitors. This new strategy was applied to detect β‐glucosidase and monitor β‐glucosidase inhibitor in hepatoma cells using cell imaging. All results suggest that the new method is sensitive and promising for use in cancer diagnosis and treatment.     

Difference in the structures of alanine tri‐ and tetra‐peptides with antiparallel β‐sheet assessed by X‐ray diffraction,solid‐state NMR and chemical shift calculations by GIPAW

Alanine oligomers provide a key structure for silk fibers from spider and wild silkworms.We report on structural analysis of l ‐alanyl‐l ‐alanyl‐l ‐alanyl‐l ‐alanine (Ala)₄ with anti‐parallel (AP) β‐structures using X‐ray and solid‐state NMR. All of the Ala residues in the (Ala)₄ are in equivalent positions, whereas for alanine trimer (Ala)₃ there are two alternative locations in a unit cell as reported previously (Fawcett and Camerman, Acta Cryst., 1975, 31, 658–665). (Ala)₄ with AP β‐structure is more stable than AP‐(Ala)₃ due to formation of the stronger hydrogen bonds. The intermolecular structure of (Ala)₄ is also different from polyalanine fiber structure, indicating that the interchain arrangement of AP β‐structure changes with increasing alanine sequencelength. Furthermore the precise ¹H positions, which are usually inaccesible by X‐ray diffraction method, are determined by high resolution ¹H solid state NMR combined with the chemical shift calculations by the gauge‐including projector augmented wave method. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 13–20, 2014.     

Zero and second‐derivative synchronous fluorescence spectroscopy for the quantification of two non‐classical β‐lactams in pharmaceutical vials: Application to stability studies

The formation of metal chelates with various ligands may lead to the production of fluorescent chelates or enhance the fluorescence of the chelating agent. This paper describes two sensitive, selective and computer‐solved methods, namely, zero order (SF) and second‐derivative synchronous spectrofluorimetry (SDSFS) for nano‐quantitation of two carbapenems; meropenem (MP) and ertapenem (EP). The methods are based on the chelation of MP with Tb³⁺ and EP with Zr⁴⁺ in buffered organic medium at pH 4.0 to produce fluorescent chelates. In the zero order method, the relative synchronous fluorescence intensity is measured at 327.0 nm at Δλ = 70.0 and 100.0 nm for MP and EP, respectively. The second method utilizes a second‐derivative technique to enhance the method selectivity and emphasize a stability‐indicating approach. The peak amplitudes (²D) of the second‐derivative synchronous spectra were estimated to be 333.06 and 330.06 nm for MP and EP, respectively. The proposed synchronous spectrofluorimetric methods were validated according to the International Conference on Harmonization (ICH) guidelines and applied successfully for the analysis of MP and EP in pure forms, pharmaceutical vials and in synthetic mixtures with different degradants of both drugs. Under optimum conditions, the mole‐ratio method was applied and the co‐ordination ratios of MP–Tb³⁺ and EP–Zr⁴⁺chelates were found to be 1:1 and 1:3. The formation constants for the chelation complexes were evaluated using the Benesi–Hildebrand's equation; the free energy change (ΔG) was also calculated. The results indicated that EP–Zr⁴⁺ was more stable than the MP–Tb³⁺ chelate. Moreover, the developed methods were found to be selective and inexpensive for quantitative determination of both drugs in quality control laboratories at nano‐levels.     

Luminescent probing of the simplest chiral α‐amino acid–alanine in an enantiopure and racemic state

Luminescent spectroscopy combined with the technique of luminescent probing with rare earth ions (europium, gadolinium, terbium) and an actinide ion (uranyl) was used to differentiate enantiopure and racemic alanine, the simplest chiral proteinogenic amino acid. Using the achiral luminescent probes, small differences between pure L and DL alanine in the solid state were strongly amplified. Based on the observed electronic transitions of the probes, the position of the triplet level of the coordinated alanine was estimated. Formation of homo‐ and heterochiral complexes between enantiomers of alanine and the metal ions is discussed as a possible mechanism of chiral self‐discrimination.     

Effect of preferential solvation and bimolecular quenching reactions on 3OCE in acetonitrile and 1,4‐dioxane binary mixtures by optical absorption and fluorescence studies

Fluorescence quenching and preferential solvation of a coumarin derivative, namely 3‐[2‐oxo‐2‐(2‐oxo‐2H‐chromen‐3‐yl)‐ethylidene]‐1,3‐dihydro‐indol‐2‐one (3OCE), with aniline used as a quencher in solvent mixtures of acetonitrile (AN) and 1,4‐dioxane (DX) was carried out at steady state. Suppan's theory of dielectric enrichment was used to understand the nonideality and dielectric enrichment in AN–DX solvent mixtures. The effect of viscosity and dielectric constant variation at room temperature were analyzed. Quenching was characterized using Stern–Volmer plots with an upward curvature. It was found that 3OCE underwent combined static and dynamic quenching that was evident from the quenching rate parameters.     

Large scale conformational transitions in β‐structural motif of gramicidin A: kinetic analysis based on CD and FT‐IR data

Gramicidin A (gA) is a polypeptide antibiotic, which forms dimeric channels specific for monovalent cations in artificial and biological membranes. It is a polymorphic molecule that adopts a unique variety of helical conformations, including antiparallel double‐stranded ↑↓β5.6 or ↑↓β7.2 helices (number of residues per turn) and a single‐stranded β6.3 helix (the ‘channel form’). The behavior of gA‐Cs⁺ complex in the micelles of TX‐100 was studied in this work. Transfer of the complex into the micelles activates a cascade of sequential conformational transitions monitored by CD and FT‐IR spectroscopy: At the first step after Cs⁺ removal, the RH ↑↓β5.6 helix is formed, which has been discussed so far only hypothetically. Kinetics of the transitions was measured, and the activation parameters were determined. The activation energies of the ↑↓β5.6 → β‐helical monomer transition in dioxane and dioxane/water solutions were also measured for comparison. The presence of water raises the transition rate constant ~10³ times but does not lead to crucial fall of the activation energy. All activation energies were found in the 20–25 kcal/mol range, i.e. much lower than would be expected for unwinding of the double helix (when 28 H‐bonds are broken simultaneously). These results can be accounted for in the light of local unfolding (or ‘cracking’) model for large scale conformational transitions developed by the P. G.Wolynes team [Miyashita O, Onuchic JN, Wolynes PG. Proc. Natl. Acad. Sci. USA 2003; 100: 12570‐12575.]. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.