Fluorescence spectroscopic studies on binding of a flavonoid antioxidant quercetin to serum albumins

Binding of quercetin to human serum albumin (HSA) was studied and the binding constant measured by following the red-shifted absorption spectrum of quercetin in the presence of HSA and the quenching of the intrinsic protein fluorescence in the presence of different concentrations of quercetin. Fluorescence lifetime measurements of HSA showed decrease in the average lifetimes indicating binding at a location, near the tryptophan moiety, and the possibility of fluorescence energy transfer between excited tryptophan and quercetin. Critical transfer distance (R _o ) was determined, from which the mean distance between tryptophan-214 in HSA and quercetin was calculated. The above studies were also carried out with bovine serum albumin (BSA).     

Conformation and Thermodynamic Properties of the Binding of Vitamin C to Human Serum Albumin

The binding of vitamin C, L-ascorbic acid (AsA), with human serum albumin (HSA) was investigated by various spectroscopic techniques under simulated physiological conditions. The fluorescence quenching constants (K _SV) at four different temperatures (292, 298, 304, and 310 K) were obtained. The thermodynamic parameters ΔH ^∘ and ΔS ^∘ were calculated to be 6.02 kJ⋅mol⁻¹ and 84.55 J⋅mol⁻¹⋅K⁻¹ using the van’t Hoff equation. Additional experiments to determine the stoichiometry (n) were carried out using isothermal titration calorimetry (ITC) and cyclic voltammetry (CV). The distance, r, between AsA and the tryptophan residues of HSA was calculated to be 3.7 nm according to F?rster’s non-radiation energy transfer theory. The effect of AsA on the conformation of HSA was studied by means of three dimensional fluorescence spectra and CD spectra. The results indicate that the presence of AsA resulted in a slight change of the HSA secondary structure. The effect of common ions on the binding of AsA to HSA was also examined.     

Study on the Interaction of Nd3+ with Human Serum Albumin at Molecular Level

Neodymium is applied widely in agriculture to improve crop nutrition and incidentally in fertilizers, yet little is known of its effect on the biological function of human serum albumin (HSA). The interaction of Nd³⁺ to HSA has been investigated mainly by fluorescence spectra, UV–vis absorption spectra and circular dichroism (CD) under simulative physiological conditions. Fluorescence data revealed that the quenching mechanism of HSA by Nd³⁺ was a static quenching process and the binding constant is 5.71 × 10⁴ L mol^‐1 and the number of binding sites is 1 at 292 K. The thermodynamic parameters (ΔH⁰ = ‐20.79 kJ mol^‐1, ΔG⁰ = ‐26.58 kJ mol^‐1, and ΔS⁰ = 19.85 J mol^‐1 K^‐1) indicate that electrostatic effect between the protein and Nd³⁺ is the main binding force. The distance r = 2.91 nm between donor (HSA) and acceptor (Nd³⁺) was obtained according to Förster's nonradiative energy transfer. In addition, UV–vis, CD and synchronous fluorescence results showed that the addition of Nd³⁺ changed the conformation of HSA.     

Studies on Thermodynamics Features of the Interaction between Imidacloprid and Bovine Serum Albumin

At different temperatures, the interactions between imidacloprid （IMI） and bovine serum albumin （BSA） were investigated with a fluorescence quenching spectrum, a synchronous fluorescence spectrum, a three-dimensional fluorescence spectrum and an ultraviolet-visible spectrum. The average values of bonding constants （KLB： 3.424 × 10^4 L,mol^-1）, thermodynamic parameters （△H： 5.188 kJ,mol^-1, △G^（○—）：-26.36 kJ,mol^-1, △S： 103.9 J,K^-1,mol^-1） and the numbers of bonding sites （n： 1.156） could be obtained through Stern-Volmer, Lineweaver-Burk and ther- modynamic equations. It was shown that the fluorescence of BSA could be quenched for its reactions with IMI to form a certain kind of new compound. The quenching belonged to a static fluorescence quenching, with a non-radiation energy transfer happening within a single molecule. The thermodynamic parameters agree with △H〉 0, △S〉0 and△G^（○-）〈0, suggesting that the binding power between IMI and BSA should be mainly a hydrophobic interaction.     

Fluorescence resonance energy transfer from tryptophan in human serum albumin to a bioactive indoloquinolizine system

The interaction between a bioactive molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with human serum albumin (HSA) has been studied using steady-state absorption and fluorescence techniques. A 1:1 complex formation has been established and the binding constant (K) and free energy change for the process have been reported. The AODIQ-HSA complex results in fluorescence resonance energy transfer (FRET) from the tryptophan moiety of HSA to the probe. The critical energy-transfer distance (R ₀) for FRET and the Stern-Volmer constant (K _sv) for the fluorescence quenching of the donor in the presence of the acceptor have been determined. Importantly, K _SV has been shown to be equal to the binding constant itself, implying that the fluorescence quenching arises only from the FRET process. The study suggests that the donor and the acceptor are bound to the same protein at different locations but within the quenching distance.     

Interaction Between Ginkgolic Acid and Human Serum Albumin by Spectroscopy and Molecular Modeling Methods

The interaction of ginkgolic acid (15:1, GA) with human serum albumin (HSA) was investigated by FT–IR, CD and fluorescence spectroscopic methods as well as molecular modeling. FT–IR and CD spectroscopic showed that complexation with the drug alters the protein’s conformation by a major reduction of α-helix from 54 % (free HSA) to 46–31 % (drug–complex), inducing a partial protein destabilization. Fluorescence emission spectra demonstrated that the fluorescence quenching of HSA by GA was by a static quenching process with binding constants on the order of 10⁵ L·mol^?1. The thermodynamic parameters (ΔH = ?28.26 kJ·mol^?1, ΔS = 11.55 J·mol^?1·K^?1) indicate that hydrophobic forces play a leading role in the formation of the GA–HSA complex. The ratio of GA and HSA in the complex is 1:1 and the binding distance between them was calculated as 2.2 nm based on the Förster theory, which indicates that the energy transfer from the tryptophan residue in HSA to GA occurs with high probability. On the other hand, molecular docking studies reveal that GA binds to Site II of HSA (sub-domain IIIA), and it also shows that several amino acids participate in drug–protein complexation, which is stabilized by H-bonding.     

Binding Studies of a Schiff Base Compound Containing a 1,2,4-Triazole Ring with Bovine Serum Albumin Using Spectroscopic Methods

The binding interaction of a Schiff base compound containing a 1,2,4‐triazole ring [4‐(4‐chlorobenzyl‐ideneamino)‐5‐methyl‐1,2,4‐triazole‐3‐thiol, CTT] with bovine serum albumin (BSA) was studied by spectroscopy methods including fluorescence and circular dichroism spectrum under simulative physiological conditions. Fluorescence investigation revealed that the fluorescence quenching of BSA was induced by the formation of a relative stable CTT‐BSA complex. The corresponding binding constants (K_a) between CTT and BSA at three different temperatures were calculated according to the modified Stern‐Volmer equation. The enthalpy change (ΔH^⊖) and entropy change (ΔS^⊖) were calculated to be −15.78 kJ·mol⁻¹ and 49.23 J·mol⁻¹·K⁻¹, respectively, which suggested that hydrophobic forces and hydrogen bond played major roles in stabilizing the CTT‐BSA complex. Site marker competitive experiments indicated that the binding of CTT to BSA primarily took place in sub‐domain IIIA (site II) of BSA. The binding distance (r) between CTT and the tryptophan residue of BSA was obtained to be 4.3 nm based on F?rster theory of non‐radioactive energy transfer. The conformational investigation revealed that the presence of CTT decreased the α‐helix content of BSA (from 58.62% to 54.66%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro‐environmental and conformational changes of BSA molecules.     

Studies of Interaction between Ergosta‐4,6,8(14),22‐tetraen‐3‐one (Ergone) and Human Serum Albumin by Molecular Spectroscopy and Modeling

Our previous experimental results have shown that ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) is one of the main bioactive components of Polyporus umbellatus. The efficacy of ergone binding to human serum albumin (HSA) is critical for pharmacokinetic behavior of ergone. The interactions between ergone and HSA under simulative physiological conditions were investigated by the methods of fluorescence spectroscopy, absorption and circular dichroism spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by ergone was the result of the formation of the ergone‐HSA complex. According to the modified Stern‐Volmer equation, the binding constants (K_a) between ergone and HSA were determined. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be 0.989 kJ mol^‐1 and 11.214 J mol^‐1 K^‐1, indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding of ergone to HSA. The conformational investigation showed that the presence of ergone decreased the α‐helical content of HSA and induced the slight unfolding of the polypeptides of protein. Furthermore, displacement experiments using warfarin and ibuprofen indicated that ergone could bind to site I of HSA, which was also in agreement with the results of the molecular modeling.     

Determination of the Interaction of Arsenic and Human Serum Albumin by Online Microdialysis Coupled to LC with Hydride Generation Atomic Fluorescence Spectroscopy

Study on the stoichiometry and affinity of the arsenicals bound to HSA is an important step toward a better understanding of arsenic toxic effects. After incubation of As^III or As^V with HSA at the physiological conditions (pH 7.43 and 37 °C), the free arsenicals and arsenic-HSA complexes were separated and detected by the combined techniques of microdialysis and liquid chromatography with hydride generation atomic fluorescence spectroscopy (MD–LC–HGAFS). The decrease of As^III peak response rather than As^V indicated that HSA reacted with As^III but not As^V. The binding plots indicated that the binding between HSA and As^III was in Scatchard pattern when the concentration ratios of As^III to HSA were ≤1:1. The strong binding sites (n ₁) were 1.6 and the stability constant (K ₁) was 1.54 × 10⁶ M⁻¹. When the concentration ratios of As^III to HSA were >1:1, the binding was in Plasvento pattern with the stability constant K ₂ ≅ 0 and no specific binding of As^III with HSA. On the contrary, As^V did not show binding with HSA. The results showed that As^III reacted with HSA more readily than As^V, which provides a chemical basis for arsenic toxicity.

     

Insight into the binding evaluation of two antitumor Pd(II) complexes with human serum albumin

The interaction between two novel water-soluble palladium(II) complexes (Pd(bpy)(pyr-dtc)]NO₃, complex I and ([Pd(phen)(pyr-dtc)]NO₃, complex II, where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and pyr-dtc = pyrrolidinedithiocarbame) and human serum albumin (HSA) was investigated by fluorescence quenching spectroscopy, synchronous, fluorescence resonance energy transfer (FRET) and three-dimensional fluorescence combined with UV–Vis absorption spectroscopy and circular dichroism technique under simulative physiological conditions. Fluorescence analysis demonstrated that the quenching mechanism of HSA by Pd(II) complexes was static fluorescence quenching and hydrogen bonds and van der Waals interactions were the main intermolecular force based on thermodynamic data. The HSA–Pd(II) complex interaction had a high affinity of 10⁵ M^?1, and the number of binding sites n is almost 1. The results of synchronous fluorescence, three-dimensional fluorescence spectra, UV–Vis absorption and CD spectroscopy indicated that these two complexes may induce the microenvironment around the tryptophan residues and the conformation of human serum albumin. The binding distance (r) in the interaction between Pd(II) complex and HSA was estimated by the efficiency of fluorescence resonance energy transfer (FRET). Furthermore, results from multiple spectroscopic studies are consistent and indicate that the antitumor Pd(II) complexes can efficiently bind with human serum albumin molecules, providing a reasonable model that can help in understanding the design, transportation and toxic effects of anticancer agents.     

Theoretical Study on Electron Transfer Matrix Element in Oxidation of α—Amino Carbon—Centered Radical by O2

As a successive work of our previous paper,^1the electron transfer matrix element(Vrp)in the oxidation of the simplified model molecule of α-amino carbon-centered radical by O2 has been investigated with ab initio calculation at the level of UHF/6-31 G**.Based on the optimized geometries of the reactgant and the ion-pair complex obtained previously,the reaction heat and the iuner reorganization energy have been obtained by constructing the potential energy curves of reactant and product states considering the solvent effect with the conductor-like screening model(COSMO).The solvent reorganization energy has been estimated using Lippert-Mataga relationship.The calculated results show that the value of Vrp is several times larger than that of RT,which means that the model reaction is an adiabatic one.Theoretical investigation indicates that the solvent effect on the direct electron transfer (ET) process of oxidation of α-amino carbon-centered radical by oxygen is remarkable.     

A Spectroscopic Survey of Substituted Indoles Reveals Consequences of a Stabilized 1Lb Transition

Although tryptophan is a natural probe of protein structure, interpretation of its fluorescence emission spectrum is complicated by the presence of two electronic transitions, ¹L_a and ¹L_b. Theoretical calculations show that a point charge adjacent to either ring of the indole can shift the emission maximum. This study explores the effect of pyrrole and benzyl ring substitutions on the transitions' energy via absorption and fluorescence spectroscopy, and anisotropy and lifetime measurements. The survey of indole derivatives shows that methyl substitutions on the pyrrole ring effect ¹L_a and ¹L_b energies in tandem, whereas benzyl ring substitutions with electrophilic groups lift the ¹L_a/¹L_b degeneracy. For 5‐ and 6‐hydroxyindole in cyclohexane, ¹L_a and ¹L_b transitions are resolved. This finding provides for ¹L_a origin assignment in the absorption and excitation spectra for indole vapor. The 5‐ and 6‐hydroxyindole excitation spectra show that despite a blue‐shifted emission spectrum, both the ¹L_a and ¹L_b transitions contribute to emission. Fluorescence lifetimes of 1⁰ ns for 5‐hydroxyindole are consistent with a charge acceptor‐induced increase in the nonradiative rate (1).     

Spectroscopic and Molecular Docking Approaches for Investigation of Interaction of Phellopterin with Human Serum Albumin

The mechanism of interaction between human serum albumin (HSA) and natural product phellopterin (PL) from Angelica dahurica was investigated by spectroscopic techniques with molecular docking under simulated physiological conditions. The experimental results showed that the fluorescence of HSA was regularly quenched by PL, and the quenching constants (K_SV) decreased with increasing temperature, which indicated that the quenching mechanism was a static quenching procedure. The binding constants (K_A) were larger than 10^?5 M^?1 and the number of binding sites (n) was approximate to 1 at different temperatures, which indicated that the binding affinity was hige and there was just one main binding site in HSA for PL. According to thermodynamic parameters from Van't Hoff equation, the binding process of PL with HSA was spontaneous and exothermic process due to ΔG < 0, and the electrostatic force played major role in the binding between PL and HSA according to ΔH < 0 and ΔS > 0. The binding distance (r) was calculated to be about 3.35 nm, which implied that the energy transfer from HSA to PL occurred with high possibility according to the theory of Förster's non-radiation energy transfer. The microenvironment and conformation of HSA changed with the addition of PL based on the results of synchronous and three-dimensional fluorescence methods. The molecular docking analysis revealed the binding locus of PL to HSA in subdomain IIIA (Sudlow's site II).     

Synthesis,crystal structures and DNA/human serum albumin binding of ternary Cu(II) complexes containing amino acids and 6‐(pyrazin‐2‐yl)‐1,3,5‐triazine‐2,4‐diamino

Two water‐soluble 6‐(pyrazin‐2‐yl)‐1,3,5‐triazine‐2,4‐diamino (pzta)‐based Cu(II) complexes, namely [Cu(l ‐Val)(pzta)(H₂O)]ClO₄ ( 1 ) and [Cu(l ‐Thr)(pzta)(H₂O)]ClO₄ ( 2 ) (l ‐Val: l ‐valinate; l ‐Thr: l ‐threoninate), were synthesized and characterized using elemental analyses, molar conductance measurements, spectroscopic methods and single‐crystal X‐ray diffraction. The results indicated that the molecular structures of the complexes are five‐coordinated and show a distorted square‐pyramidal geometry, in which the central copper ions are coordinated to N,N atoms of pzta and N,O atoms of amino acids. The interactions of the complexes with DNA were investigated using electronic absorption, competitive fluorescence titration, circular dichroism and viscosity measurements. These studies confirmed that the complexes bind to DNA through a groove binding mode with certain affinities (K_b = 4.71 × 10³ and 1.98 × 10³ M^?1 for 1 and 2 , respectively). The human serum albumin (HSA) binding properties of the complexes were also evaluated using fluorescence and synchronous fluorescence spectroscopies, indicating that the complexes could quench the intrinsic fluorescence of HSA in a static quenching process. The relevant thermodynamic parameters revealed the involvement of van der Waals forces and hydrogen bonds in the formation of complex–HSA systems. Finally, molecular docking technology was also used to further verify the interactions of the complexes with DNA/HSA.     

High‐Field Pulsed EPR Spectroscopy for the Speciation of the Reduced [PV2Mo10O40]6− Polyoxometalate Catalyst Used in Electron‐Transfer Oxidations

An in‐depth spectroscopic EPR investigation of a key intermediate, formally notated as [PV^IVV^VMo₁₀O₄₀]^6? and formed in known electron‐transfer and electron‐transfer/oxygen‐transfer reactions catalyzed by H₅PV₂Mo₁₀O₄₀, has been carried out. Pulsed EPR spectroscopy have been utilized: specifically, W‐band electron–electron double resonance (ELDOR)‐detected NMR and two‐dimensional (2D) hyperfine sub‐level correlation (HYSCORE) measurements, which resolved ⁹⁵Mo and ¹⁷O hyperfine interactions, and electron–nuclear double resonance (ENDOR), which gave the weak ⁵¹V and ³¹P interactions. In this way, two paramagnetic species related to [PV^IVV^VMo₁₀O₄₀]^6? were identified. The first species (30–35 %) has a vanadyl (VO²⁺)‐like EPR spectrum and is not situated within the polyoxometalate cluster. Here the VO²⁺ was suggested to be supported on the Keggin cluster and can be represented as an ion pair, [PV^VMo₁₀O₃₉]^8?[V^IVO²⁺]. This species originates from the parent H₅PV₂Mo₁₀O₄₀ in which the vanadium atoms are nearest neighbors and it is suggested that this isomer is more likely to be reactive in electron‐transfer/oxygen‐transfer reaction oxidation reactions. In the second (70–65 %) species, the V^IV remains embedded within the polyoxometalate framework and originates from reduction of distal H₅PV₂Mo₁₀O₄₀ isomers to yield an intact cluster, [PV^IVV^VMo₁₀O₄₀]^6?.     

FLUORESCENCE INVESTIGATIONS OF ALBUMIN FROM PATIENTS WITH FAMILIAL DYSALBUMINEMIC HYPERTHYROXINEMIA*

Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant syndrome in which clinically euthyroid patients have elevated total thyroxine levels. These high serum thyroxine levels are traceable to altered binding of thyroxine to the patient's albumin. Albumin from FDH patients and normal volunteers have been purified. Reverse-phase and ion-exchange high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the FDH-human serum albumin (HSA) samples show a single band that comigrates with normal HSA. In both protein solutions the intrinsic fluorescence, upon 280 nm excitation, is predominantly due to the single tryptophan residue. The quantum yield of this intrinsic fluorescence in the FDH-HSA solutions is, however, reduced relative to that of HSA. Furthermore, the “average” lifetime value of the tryptophan emission in the FDH-HSA sample is less than that of normal HSA, consistent with its reduced quantum yield. The binding of thyroxine to both albumins effectively quenches the tryptophan emission probably via a nonradiative energy transfer mechanism. Time-resolved data suggest that the albumin from the dysalbuminemic patients is actually an approximately equimolar mixture of normal HSA and FDH-HSA indicative of heterologous expression. Quenching of the intrinsic HSA and FDH-HSA fluorescence by serial additions of thyroxine showed enhanced quenching of FDH-HSA relative to HSA at any T4 to albumin mole ratio, therefore supporting earlier reports of increased thyroxine affinity to FDH-HSA.     

A mononuclear nickel(II) complex based on polypyridyl ligand: synthesis,characterization, and biological activity

[Ni(acac)₂(o-NPIP)](CH₃OH)₃ (acac = acetylacetonate), based on the polypyridyl ligand 2-(2-nitrophenyl)imidazo[4,5-f]1,10-phenanthroline) (o-NPIP), has been synthesized and characterized by single-crystal analysis, IR and electronic spectra. In the structure of Ni(II) complex, the coordination sphere around Ni(II) is distorted octahedral with one o-NPIP and two acetylacetonates. DNA binding and human serum albumin (HSA) interactions with the Ni(II) complex have been investigated by electronic absorption and fluorescence measurements, revealing that the Ni(II) complex binds with DNA via intercalative binding. The quenching constants verified a dynamic quenching mechanism between HSA and the Ni complex by fluorescence quenching. ΔG, ΔH, and ΔS at different temperatures (288, 298, and 310 K) indicated that hydrophobic interactions play a major role. Synchronous fluorescence spectral experiments revealed that the Ni(II) complex affected the microenvironment around the tryptophan residue of HSA.     

A Highly Selective and Turn‐on Fluorescent Probe for Fe3+ Ion Based on Perylene Tetracarboxylic Diimide

We have demonstrated a turn‐on fluorescent sensor 6 for detection of Fe³⁺ based on photo‐induced electron transfer (PET) mechanism. The probe comprises a perylene tetracarboxylic diimide (PDI) fluorophore and two bis((1,2,3‐triazol‐4‐yl)methyl)amine (DTA) moieties as the metal ion receptors. It exhibits high selectivity toward Fe³⁺ over various other metal ions in CH₃CN/H₂O (1:1, V/V). The binding stoichiometry for 6 ‐Fe³⁺ complexes has been determined to be 1:2 by a Job plot of fluorescence. The association constant between 6 and Fe³⁺ was estimated to be 1.04×10¹⁰ (mol/L)^?2 by Benesi‐Hildebrand equation.     

Superoxide Dismutase Activity of the Naturally Occurring Human Serum Albumin–Copper Complex without Hydroxyl Radical Formation

The superoxide radical anion (O₂^.?) is biologically toxic and contributes to the pathogenesis of various diseases. Here we describe the superoxide dismutase (SOD) activity of human serum albumin (HSA) complexed with a single Cu^II ion at the N‐terminal end (HSA–Cu complex). The structure of this naturally occurring copper‐coordinated blood serum protein has been characterized by several physicochemical measurements. The O₂^.? dismutation ability of the HSA–Cu (1:1) complex is almost the same as that of the well‐known SOD mimics, such as Mn^III‐tetrakis(N‐methylpyridinium)porphyrin. Interestingly, the HSA–Cu complex does not induce a subsequent Fenton reaction to produce the hydroxyl radical (OH^.), which is one of the most harmful reactive oxygen species.     

ENERGY TRANSFER STUDIES ON CRYPTOMONAD BILIPROTEINS

Abstract. Fluorescence techniques of various types have been used to study the light-gathering and energy transfer modes for various cryptomonad biliproteins (phycocyanin or phycoerythrins). Analysis of fluorescence polarization and absorption data demonstrates that each cryptomonad biliprotein is composed of at least two distinct types of absorbing chromophore, each attached to the protein through covalent linkages to different polypeptide chains. Examination of the fluorescence emission spectra as a function of excitation at several wavelengths demonstrates that only one of these absorbing chromo-phores is responsible for the fluorescence. This behavior is consistent with a known phenomenon whereby photons are gathered by more than one chromophore and then after radiationless energy transfer are emitted by only one chromophore. Application of Förster dipole-dipole energy transfer theory is made to the study of the mode by which energy absorbed by biliproteins migrates to Chl a. The spectral overlap integral between phycocyanin (Chroomonas sp.) and Chl a is 7.13 ± 10^-10cm⁶mol^-1and between phycocyanin and Chl c₂ 0.25 ± 10^-10cm⁶mol^-1. This large difference in overlap suggests, although does not prove, that phycocyanin might transfer energy directly to Chl a without a Chl c₂ intermediary. The cryptomonad phycoerythrins may also use this method but a Chl c₂ intermediate could not be ruled out for them. Radiationless energy transfer among homogeneous biliproteins is shown to be feasible. All these calculations are based on in vitro spectra and the interpretations extrapolated to the cellular situation, and these tentative conclusions are reached without knowledge of other factors, such as chromophore-chro-mophore orientation and distance, which could greatly influence the energy transfer scheme.