Gadolinium and calcium binding to bovine serum albumin

The temperature jump relaxation technique is used to study the binding of calcium(II) and gadolinium(III) to bovine serum albumin at 7°C and an ionic strength of 0·2 (NaClO₄). Both metal ions are predicted to bind to the ϵ-amino groups of the protein above pH = 8·2, where extensive metal ion hydrolysis takes place. For the reaction between GdOH²⁺ and bovine serum albumin, the complex formation rate constant is (1·0±0·1) × 10⁸ M⁻¹ sec⁻¹ and the dissociation rate constant is (9·3±2·1) sec⁻¹. The corresponding calcium association reaction is too rapid to obtain rate constants. The complex formation rate constant is compared to other gadolinium systems to explain the relatively high complexation constant with bovine serum albumin.     

Betulinic acid binding to human serum albumin: a study of protein conformation and binding affinity

Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be K(BA)=1.685+/-0.01 x 10(6) M(-1), indicating a strong binding affinity. The secondary structure of the HSA-BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA+drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies.     

Capillary electrophoresis study of human serum albumin binding to basic drugs

Summary The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm × 50 μm i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients,r>0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.     

Polyoxometalate binding to human serum albumin: a thermodynamic and spectroscopic approach

The molecular recognition of polyoxometalates by human serum albumin is studied using two different polyoxometalates (POMs) at pH 7.5. The results are compared with those obtained at pH 3.5 and 9.0. At pH 7.5, both POMs strongly interact with the protein with different binding behaviors. The Keggin shaped POM, [H(2)W(12)O(40)](6-) (H2W12), specifically binds the protein, forming a complex with a 1:1 stoichiometry with Ka = 2.9 x 10(6) M(-1). The binding constant decreased dramatically with the increase of the ionic strength, thus indicating a mostly electrostatic binding process. Isothermal titration calorimetry (ITC) experiments show that the binding is an enthalpically driven exothermic process. For the wheel shaped POM [NaP(5)W(30)O(110)](14-) (P5W30), there are up to five binding sites on the protein. Increasing the ionic strength changes the binding behavior significantly, leading to a simple exothermic process, with several binding sites. Competitive binding experiments indicate that the two POMs share one common binding site. In addition, they show the existence of another important binding site for P5W30. The two POMs exhibit different binding dependences on the pH. The combination of the experimental results with the knowledge of the surface map of the protein in its N-B conformation transition domain leads to the proposal for the probable binding site of POMs. The present work reveals a protein conformation change upon P5W30 binding, a new feature not explicitly documented in previous studies.     

Spectroscopic studies on binding of Puerarin to human serum albumin

The interaction between Puerarin with human serum albumin has been studied for the first time by spectroscopic methods including fluorescence quenching technology, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that Puerarin can strongly quench the intrinsic fluorescence of HSA by static quenching and there is a single class of binding site on HSA. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the binding of Puerarin to HSA changed slightly molecular conformation of HSA. Furthermore, the thermodynamic functions ΔH⁰ and ΔS⁰ for the reaction were calculated to be −9.067 kJ mol⁻¹ and 54.315 J mol⁻¹ K⁻¹ according to van’t Hoff equation. These data suggested that both hydrogen bond and hydrophobic interaction play a major role in the binding of Puerarin to HSA, which is in good agreement with the result of molecular modeling study.     

High-performance liquid chromatographic determination of the binding of ceftriaxone to human serum albumin solution and albumin from diluted human serum

The binding of ceftriaxone to human serum albumin has been studied by high-performance liquid chromatography. The gel permeation method of Hummel and Dreyer was used. Ceftriaxone was tested with two sources of albumin (aqueous solution and diluted serum). After internal calibration the binding parameters were determined for each albumin, and results compared. These data are in agreement with those from classical methods for the determination of protein binding of ceftriaxone.     

Retention of structurally diverse drugs in human serum albumin chromatography and its potential to simulate plasma protein binding

The retention behavior of 39 structurally diverse neutral, basic and acidic drugs was investigated on an HSA stationary phase using PBS buffer (pH 7.0) and acetonitrile or 2-propanol as organic modifiers. Extrapolated or directly measured log k_w values as well as isocratic retention factors were correlated with plasma protein binding data taken from the literature. Retention factors determined in the presence of 10% acetonitrile led to high quality 1:1 correlation with apparent log K_HSA values. The derived reference equation was successfully validated using a secondary set of 24 drugs. Further analysis of HSA retention into more fundamental properties revealed the involvement of anionic species in solute-stationary phase interactions, expressed by the negatively charged fraction, besides the partitioning mechanism which was reflected by lipophilicity. Protonation of basic drugs, although less important, may also influence retention, leading to reduced partitioning into the HSA surface as a net effect, while it seems to have no effect on HSA binding. The above results were further confirmed by linear solvation energy relationships (LSER).     

Induced chirality upon crocetin binding to human serum albumin: origin and nature

Binding to human serum albumin (HSA) of the natural, achiral carotenoid crocetin, having hypocholesterolemic and antitumour effects, was investigated in detail by circular dichroism (CD) and absorption spectroscopy. It has been shown that in the visible absorption region the crocetin–HSA complex exhibits a well-defined induced circular dichroic spectrum with two major bands of opposite sign, proving excitonic interaction between carotenoids bound in a left-handed chiral arrangement on the albumin molecule. In the course of CD titration experiments, palmitic acid gradually decreased the exciton band intensities indicating that crocetin and palmitic acid have common binding sites on HSA. To investigate potential sources of the intermolecular excitonic interaction, molecular modeling studies were performed fitting crocetin molecules to the long-chain fatty acid binding sites of HSA, determined recently by X-ray crystallographic measurements. The results suggest that binding of crocetin to domain III of the albumin might be responsible for the observed intermolecular exciton coupling. Crocetin binding was accompanied by a significant red shift in the visible absorption spectrum which has showed no excitonic contribution but rather indicates the higher polarizability of the protein environment.     

HPLC study of the binding of sulfinpyrazone to serum albumin

Summary A thin layer chromatographic method for a qualitative screening-test and a quantitative analysis of 2,4,6-TNT, biodegradation products, octogen and hexogen in ammunition wastes was developed using both polar and non-polar modified sorbents. For enrichment a solidphase extraction on LiChrolut® EN followed by removal with methanol/acetonitrile (11 v/v) was chosen. To imitate real samples, spiked tap water samples of known composition were used.     

高效亲和色谱法测定丹皮酚与固定化人血清白蛋白结合域

利用亲和色谱,在模拟人体生理环境下(37 ℃、pH 7.4),采用竞争置换法研究了丹皮酚(PAE)与固定化人血清白蛋白(HSA)的相互作用。通过对PAE的自我竞争分析及PAE与HSA上结合位点的标记物间的竞争置换分析,得到了PAE和HSA间的结合常数、结合位点数和结合域。结果表明: PAE在HSA分子中仅存在一类结合位点,结合常数为4.84×103 L/mol,该结合位点为HSA上的Sudlow siteII;通过对PAE与HSA相互作用的热力学研究,推断出二者间的作用力类型为氢键或范德华力。     

A thermodynamic study on the binding of theophylline with human serum albumin

The thermodynamic parameters of interaction between theophylline and Human Serum Albumin (HSA) in buffer solution (30 mM) of pH = 7 at 27 °C was investigated by isothermal titration calorimetry (ITC). The thermodynamic quantities of the binding mechanism, the number of binding sites (g), the dissociation binding constant (K _d), the molar enthalpy of binding (ΔΗ) and other thermodynamic parameters can be obtained by the extended solvation theory.     

Investigation of ketoprofen binding to human serum albumin by spectral methods

The binding of ketoprofen with human serum albumin (HSA) was studied by fluorescence and absorption spectroscopic methods. Quenching of fluorescence of HSA was found to be a static quenching process. At 288.15, 298.15, 308.15 and 318.15 K, the binding constants and binding sites were obtained. The effects of Cu²⁺, Al³⁺, Ca²⁺, Pb²⁺ and K⁺ on the binding at 288.15 K were also studied. The thermodynamic parameters, ΔH, ΔG and ΔS were got and the main sort of acting force between ketoprofen and HSA was studied. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r, between the acceptor (ketoprofen) and the donor (HSA) was calculated.     

Equilibrium saturation chromatographic method for studying the binding of ligands to human serum albumin by high-performance liquid chromatography. Influence of fatty acids and sodium dodecyl sulphate on warfarin-human serum albumin binding

A size exclusion chromatographic method for studying ligand-macromolecule binding parameters is described. This equilibrium saturation method allows the determination of the concentrations of constituents in equilibrium and is specially useful for characterizing ligand--protein binding under conditions that can be compared with physiological conditions. The method has been used for measuring warfarin--human serum albumin (HSA) binding and for studying the influence of free fatty acids (FFA) and sodium dodecyl sulphate on warfarin--HSA binding. Some comparisons with the Hummel and Dreyer method are given. The influence of the FFA is strongly dependent on their chain length, with an inversion of the effect for a 10-carbon chain.     

A novel lophine-based fluorescence probe and its binding to human serum albumin

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510 nm. The binding constant and binding number determined by Scatchard plot was 3.65 × 10⁶ M⁻¹ and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.     

Temperature-dependent simultaneous ligand binding in human serum albumin

Human serum albumin (HSA) is a soluble protein in our circulatory system, which is known to bind a variety of drugs and ligands. Since Sudlow's pioneering works on the ligand-binding sites, a major effort of the biophysical/biochemical research has been directed to characterize the structural, functional, and dynamical properties of this protein. Structural studies on HSA have revealed distinct temperature-induced folded states. Despite knowing about the ligand-binding properties and residues important for the binding, less is understood about the temperature-dependent molecular recognition of the protein. Here, we have prepared thermally induced unfolded states of the protein and characterized those by circular dichroism (CD) and differential thermal analysis (DTA) techniques. The change in the globular structure of the protein as a consequence of thermal unfolding has also been characterized by dynamic light scattering (DLS) measurements. We have used two fluorescent ligands (4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl) 4H-pyran) (DCM; hydrophobic; neutral) and Nile blue (NB; cationic) of different natures to characterize the ligand-binding properties of the protein in the native and thermally unfolded states. The possible binding sites of the ligands have been characterized by competitive binding with other drug molecules having definite binding sites in HSA. Picosecond-resolved F?rster resonance energy transfer (FRET) studies along with steady-state and polarization-gated spectroscopies on the ligands in the protein reveal the dynamics of the binding sites at various temperatures. From the FRET studies, an attempt has been made to characterize the simultaneous binding of the two ligands in various temperature-dependent folded states of HSA.     

Effect of pH and small inorganic ions on binding of sulfadimethoxine and sulfaphenazole to human serum albumin measured by circular dichroism

The binding of sulfadimethoxine and sulfaphenazole to human serum albumin (HSA) has been shown by circular dichroism measurements to be dependent on the N-B transition. The secondary drug binding sites were found to be optically active in the B conformation form in HSA but optically inactive in the N form. Moreover, the drug-HSA interaction in Tris-HCl buffer seems to be more sensitive to the conformational change in HSA, compared with that in the phosphate buffer.