Interaction of aspirin and vitamin C with bovine serum albumin

Vitamin C (L-ascorbic acid) has a major biological role as a natural antioxidant. Aspirin belongs to the nonsteroidal anti-inflammatory drugs and functions as an antioxidant via its ability to scavenge-OH radicals. Bovine serum albumin (BSA) is the major soluble protein constituent of the circulatory system and has many physiological functions including transport of a variety of compounds. In this report, the competitive binding of vitamin C and aspirin to bovine serum albumin has been studied using constant protein concentration and various drug concentrations at pH 7.2. FTIR and UV-Vis spectroscopic methods were used to analyze vitamin C and aspirin binding modes, the binding constants and the effects of drug complexation on BSA stability and conformation. Spectroscopic evidence showed that vitamin C and aspirin bind BSA via hydrophilic interactions (polypeptide and amine polar groups) with overall binding constants of K(vitamin C-BSA)=1.57×10(4)M(-1) and K(aspirin-BSA)=1.15×10(4)M(-1); assuming that there is one drug molecule per protein. The BSA secondary structure was altered with major decrease of α-helix from 64% (free protein) to 57% (BSA-vitamin C) and 54% (BSA-aspirin) and β-sheet from 15% (free protein) to 6-7% upon drug complexation, inducing a partial protein destabilization.     

Thyroxine binding properties of glycosylated bovine serum albumin.

Thyroid hormone, thyroxine (T4) binding properties of glycosylated bovine serum albumin (G-BSA), and intact BSA were studied by the fluorescence method. The apparent binding constants for intact BSA were 0.8 (0.16) x 10(6) M-1 at pH 5.0 and 2.18 (0.06) x 10(6) M-1 at pH 9.5 at 25 degrees C. T4 binding for G-BSA was independent of pH and the apparent binding constant was 1.4 x 10(6) M-1. Thermodynamic parameters were also evaluated from the Van't Hoff plots of the apparent binding constants at pH 7.4 and 8.5. At both pH's, the free energy, enthalpy and entropy changes were almost the same for both G-BSA and BSA.     

Interaction of water-soluble amino acid Schiff base complexes with bovine serum albumin: fluorescence and circular dichroism studies

Fluorescence spectroscopy in combination with circular dichroism (CD) spectroscopy were used to investigate the interaction of water-soluble amino acid Schiff base complexes, [Zn(L1,2)(phen)] where phen is 1,10-phenanthroline and H2L1,2 is amino acid Schiff base ligands, with bovine serum albumin (BSA) under the physiological conditions in phosphate buffer solution adjusted to pH 7.0. The quenching mechanism of fluorescence was suggested as static quenching according to the Stern-Volmer equation. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between amino acid Schiff base complexes and BSA. The thermodynamic parameters DeltaG, DeltaH and DeltaS at different temperatures (298, 310 and 318K) were calculated. The results indicate that the hydrophobic and hydrogen bonding interactions play a major role in [Zn(L1)(phen)]-BSA association, whereas hydrophobic and electrostatic interactions participate a main role in [Zn(L2)(phen)]-BSA binding process. Binding studies concerning the number of binding sites and apparent binding constant Kb were performed by fluorescence quenching method. The distance R between the donor (BSA) and acceptor (amino acid Schiff base complexes) has been obtained utilizing fluorescence resonant energy transfer (FRET). Furthermore, CD spectra were used to investigate the structural changes of the BSA molecule with the addition of amino acid Schiff base complexes. The results indicate that the interaction of amino acid Schiff base complexes with BSA leads to changes in the secondary structure of the protein. Fractional contents of the secondary structure of BSA (f(alpha), f(beta), f(turn) and f(random)) were calculated with and without amino acid Schiff base complexes utilizing circular dichroism spectroscopy. Our results clarified that amino acid Schiff base complexes could bind to BSA and be effectively transported and eliminated in the body, which could be a useful guideline for further drug design.     

Mechanistic investigation on binding interaction of bioactive imidazole with protein bovine serum albumin--a biophysical study

The interaction between bioactive imidazole derivative (PPP) and bovine serum albumin (BSA) was investigated using fluorescence and UV-vis spectral studies. The experimental results showed that the fluorescence quenching of BSA by imidazole derivative was the result of the formation of BSA-PPP complex and the effective quenching constants (K(SV)) were 2.66×10(4), 2.56×10(4), and 2.10×10(4) at 301, 310 and 318 K, respectively. Static quenching and non-radiative energy transfer were confirmed to the result in the fluorescence quenching. The binding site number n, apparent binding constant K(A) and corresponding thermodynamic parameters (ΔG, ΔH and ΔS) were measured at different temperatures. The process of binding of PPP molecule on BSA was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased.     

Binding characteristics of dopa (3,4-dihydroxyphenylalanine) and its metabolites to bovine serum albumin as measured by ultrafiltration technique.

The interaction between 3,4-dihydroxyphenylalanine (DOPA), dopamine, 3-methoxytyramine and homovanillic acid and bovine serum albumin (BSA) was investigated by the ultrafiltration technique. The apparent binding constants were determined assuming the equivalence and independence of the binding sites on the BSA molecule. The binding constants were in the range of log K = 2.85 to 3.77 with 1 to 2 binding sites. The affinity of ligands to BSA strengthened with progression of the metabolism in the order of DOPA less than dopamine less than 3-methoxytyramine less than homovanillic acid.     

L-tryptophan radical cation electron spin resonance studies: connecting solution-derived hyperfine coupling constants with protein spectral interpretations

Fast-flow electron spin resonance (ESR) spectroscopy has been used to detect a free radical formed from the reaction of l-tryptophan with Ce (4+) in an acidic aqueous environment. Computer simulations of the ESR spectra from l-tryptophan and several isotopically modified forms strongly support the conclusion that the l-tryptophan radical cation has been detected by ESR for the first time. The hyperfine coupling constants (HFCs) determined from the well-resolved isotropic ESR spectra support experimental and computational efforts to understand l-tryptophan's role in protein catalysis of oxidation-reduction processes. l-Tryptophan HFCs facilitated the simulation of fast-flow ESR spectra of free radicals from two related compounds, tryptamine and 3-methylindole. Analysis of these three compounds' beta-methylene hydrogen HFC data along with equivalent l-tyrosine data has led to a new computational method that can distinguish between these two amino acid free radicals in proteins without dependence on isotope labeling, electron-nuclear double resonance, or high-field ESR. This approach also produces geometric parameters (dihedral angles for the beta-methylene hydrogens) that should facilitate protein site assignment of observed l-tryptophan radicals as has been done for l-tyrosine radicals.     

桔皮苷与牛血清白蛋白相互作用的研究

运用荧光光谱、紫外光谱法研究了桔皮苷与牛血清白蛋白(BSA)的相互作用。桔皮苷分子与BSA作用导致BSA内源荧光猝灭,猝灭机理主要为静态猝灭,并存在非辐射能量转移。测定了不同温度下该反应的结合常数、结合位点数及结合热力学参数。结果表明:桔皮苷与BSA之间主要为氢键或范德华作用力,作用过程是一个熵增加、自由能降低的自发分子间作用过程;测得了供体与受体间结合距离r和能量转移效率E;并用同步荧光技术考察了桔皮苷对BSA构象的影响。     

A comparison study on the interaction of hyperoside and bovine serum albumin with Tachiya model and Stern–Volmer equation

The interaction between hyperoside and bovine serum albumin (BSA) was examined by fluorescence spectroscopy at 298, 304, and 310 K. The spectroscopic data were analyzed using Tachiya model and Stern–Volmer equation to determine the binding sites and apparent binding constant between hyperoside and BSA. For Tachiya model, both binding sites and apparent binding constants increased with the increasing of temperature, whereas for Stern–Volmer equation, the corresponding binding constants decreased as temperature increasing and the binding sites were independent of temperature. The positive sign of enthalpy change (ΔH) and entropy change (ΔS) suggested that hydrophobic forces played a major role in the interaction. Synchronous fluorescence spectra indicated that the conformation of protein was perturbed by the interaction of hyperoside with BSA. Moreover, the presence of metal ion affected the hyperoside-BSA binding.     

Study of the interaction of carbamazepine with bovine serum albumin by fluorescence quenching method.

The interaction between carbamazepine (CBZ) and bovine serum albumin (BSA) was studied using fluorescence spectroscopy (FS) and ultraviolet spectroscopy (UV). The experimental results showed that the CBZ could insert into the BSA and quench the inner fluorescence of BSA by forming the CBZ-BSA complex. It was found that both static quenching and non-radiation energy transfer were the main reasons leading to the fluorescence quenching. The apparent binding constants (K) between CBZ and BSA were found to be 1.8 x 10(4) (27 degrees C) and 2.8 x 10(4) (37 degrees C) and the binding site values (n) were 0.97 (27 degrees C) and 1.01 (37 degrees C), respectively. According to the Forster theory of non-radiation energy transfer, the binding distances (r) between CBZ and BSA were 3.6 nm and 3.4 nm at 27 degrees C and 37 degrees C, respectively. The process of the binding was a spontaneous molecular interaction in which entropy increased and Gibbs free energy decreased, indicating that the interaction between CBZ and BSA was mainly driven by the hydrophobic force.     

Binding of dl-tryptophan to BSA adsorbed in multilayers by polymer chains grafted onto a porous hollow-fiber membrane in a permeation mode

An anion-exchange-group-containing porous membrane in the form of a hollow fiber was prepared to immobilize bovine serum albumin (BSA) as a chiral selector. First, an epoxy-group-containing polymer chain was grafted onto the pore surface of the polyethylene porous hollow-fiber membrane pre-irradiated with an electron beam. Second, the epoxy group was converted to diethylamino and 2-hydroxyethylamino groups. Third, a BSA solution was forced to permeate through the pores of the membrane. As a result, 190 mg BSA per gram of membrane, which amounted to a degree of multilayer binding of about four, were immobilized. Subsequently, a racemic solution of tryptophan (0.02 mM) was forced to permeate through the BSA-multilayered porous membrane at a flow rate ranging from 10 to 80 ml/h. A two-stage stepwise concentration change of tryptophan in the effluent was observed due to independent chiral recognition of d- and l-tryptophan by BSA adsorbed in multilayers within the graft chains.     

光谱法研究氨基己酸与牛血清白蛋白的相互作用

利用荧光光谱、紫外-可见吸收光谱及圆二色（CD）光谱研究了模拟生理条件下的氨基己酸(ACA)与牛血清白蛋白(BSA)的相互作用。 实验结果分析表明,氨基己酸对BSA的内源性荧光具有猝灭作用,属于动态猝灭过程。 计算了2种温度下ACA-BSA体系的结合常数、结合位点数及反应的热力学参数ΔG、ΔH和ΔS分别约为-21.00 kJ/mol、-0.64 kJ/mol和-72.00 kJ/(mol·K),由此推出了二者主要通过氢键和范德华力形成摩尔比为1∶1的复合物。 依据Forster非辐射能量转移理论求得二者之间的结合距离为2.3 nm。 位点取代实验指出氨基己酸主要结合在位点Site I。 CD光谱表明,氨基己酸诱导了BSA分子二级结构微变。     

Molecular complexation of uracil with bovine serum albumin and its complex with bilirubin studied by spectroscopic methods

The interactions of uracil (U) with bovine serum albumin (BSA) and its complex with bilirubin (BR·BSA) in phosphate buffer at pH 7.4 were studied by fluorescence and electronic spectroscopy. The parameters of the resulting intermolecular complexes (binding constants, quenching rate constants, the radius of the quenching sphere-of-action, etc.) were determined. The interaction of BSA with U occurs through a static quenching of protein fluorescence and has a predominantly hydrophobic character. The effect of U on the conformational changes of the protein molecule was analyzed by synchronous fluorescence spectroscopy. Uracil binds to BR·BSA more efficiently than to the free protein due to the interaction of U with the tetrapyrrole pigment incorporated in the macromolecular complex.     

川陈皮素与牛血清白蛋白相互作用的光谱研究

用多种光谱技术研究了生理条件下川陈皮素(NOB)与牛血清白蛋白(BSA)的相互作用及热力学特征.结果表明,NOB与BSA有较强的作用,NOB能使BSA的内源荧光猝灭,并以静态猝灭为主.按照Stern-Volmer方程和双对数方程分别得出不同温度下,以及不同pH值时NOB与BSA的结合常数和结合位点数.运用紫外光谱获得常温下NOB与BSA的结合常数与荧光光谱测定值相近,热力学参数△H、△S分别为55.91 kJ·5moL~(-1)、274.61 J·5moL~(-1)·5K-1,表明其主要作用力为疏水力,NOB与BSA作用为非辐射能量转移机制,其能量转移效率与结合距离分别为0.27和1.76 nm,用参比法得出BSA荧光量子产率为0.074.同步荧光光谱研究发现川陈皮素对牛血清白蛋白构象几乎没有影响.     

纤维素纤维接枝β-环糊精对苯二酚类分子的包络识别性能

用分光光度滴定法在不同温度、不同pH值条件下测定了纤维素纤维接枝β-环糊精与苯二酚类分子形成超分子包合物的表观热力学参数. 化学计量法表明, 纤维素纤维接枝β-环糊精上环糊精与客体苯二酚类分子形成了1∶1的超分子包合物. 从主-客体间包合物的表观热力学参数、尺寸关系、溶液pH值、包合时间等因素讨论了纤维上环糊精对客体苯二酚类分子的分子识别机制. 结果表明, 纤维素纤维接枝β-环糊精上环糊精对苯二酚类分子不同位置羟基具有分子选择性和识别能力.     

Interaction of albumin with polysaccharides containing ionic groups

It is very important to clarify the mechanisms of the interaction between components of organisms. In this report, the interaction between bovine serum albumin (BSA) and ionic polysaccharides were discussed. The fluorescence spectrum of tryptophan (Trp) in BSA changed as its conformation changed. On adding polysaccharide containing sulfonic acid residues (sulfonic‐type) at pH 7.4, a remarkable blue shift of the emission maximum (λ_em) of Trp was observed. This blue shift was decreased by adding NaCl. In contrast, polysaccharide containing carboxylic acid residues (carboxy‐type) scarcely changed Trp fluorescence at the same pH. At a pH lower than the isoelectric point (PI = 4.7–4.9) of BSA, a remarkable blue shift was observed not only by adding sulfonic‐type polysaccharide but also by adding carboxy‐type polysaccharide. Moreover, using the energy transfer method, in the pH region higher than the PI of BSA, carboxylic‐type polysaccharides may interact relatively weakly with the binding site II of BSA, but sulfonic‐type ones can selectively interact with binding site I weakly and with binding site II strongly. And in the pH region lower than the PI of BSA, carboxylic‐type polysaccharides interact with binding site II strongly. On the other hand, sulfonic‐type polysaccharides are bound to both binding site I and binding site II very strongly. Copyright © 1999 John Wiley & Sons, Ltd.     

Interaction of polyethyleneimine-functionalized ZnO nanoparticles with bovine serum albumin

In biological fluids, nanoparticles are always surrounded by proteins. As the protein is adsorbed on the surface, the extent of adsorption and the effect on the protein conformation and stability are dependent on the chemical nature, shape, and size of the nanoparticle (NP). We have carried out a detailed investigation on the interaction of bovine serum albumin (BSA) with polyethyleneimine-functionalized ZnO nanoparticles (ZnO-PEI). ZnO-PEI was synthesized using a wet chemical method with a core size of ~3-7 nm (from transmission electron microscopy). The interaction of BSA with ZnO-PEI was examined using a combination of calorimetric, spectroscopic, and computational techniques. The binding was studied by ITC (isothermal titration calorimetry), and the result revealed that the complexation is enthalpy-driven, indicating the possible involvement of electrostatic interaction. To investigate the nature of the interaction and the location of the binding site, a detailed domain-wise surface electrostatic potential calculation was performed using adaptive Poisson-Boltzmann software (APBS). The result shows that the protein surface can bind the nanoparticle. On binding ZnO-PEI, the protein gets destabilized to some extent, as displayed by CD (circular dichroism) and FTIR (Fourier transform infrared) spectroscopy. Chemical and thermal denaturation of BSA, when carried out in the presence of ZnO-PEI, also indicated a small perturbation in the protein structure. A comparison of the enthalpy and entropy components of binding with those derived for the interaction of BSA with ZnO nanoparticles explains the effect of hydrophilic cationic species attached on the NP surface. The effect of the NP surface modification on the structure and stability of BSA would find useful applications in nanobiotechnology.     

Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions

In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).     

Application of resonance Raman spectrometry to the determination of vitamin B12.

A gel filtration technique is described for the study of the complexation of dissolved metals by humic and fulvic acids. Measurements can be made under realistic conditions of pH and free metal ion concentrations, both of which can be maintained by the use of TRIS as a buffer. The procedure permits the determination not only of the overall stability constant, but of the binding stoichiometries and the intrinsic stability constants associated with the various types of metal binding sites. The procedure has been applied to the investigation of the interaction of fresh-water fulvic acids with Cu, Zn and Ni, and has provided evidence for the existence of two different types of binding sites in the fulvic acid molecule.     

可生物降解肝素钠/两性壳聚糖复合物用于蛋白药物pH响应释放研究

制备了一类可生物降解肝素钠两性壳聚糖复合物(HPACS),并探索将其用于蛋白药物pH响应释放.两性壳聚糖由壳聚糖与丙烯酸加成反应得到,丙烯酸取代度可通过丙烯酸壳聚糖投料比调控;用胶体与pH浊度滴定研究了肝素钠与两性壳聚糖的复合作用,发现两组分在一定pH范围内能通过静电相互作用形成复合物,复合转变临界pH(pHΦ)与两性壳聚糖中丙烯酸取代度有关,取代度越低,pHΦ值越高.以牛血清白蛋白(BSA)为模型,测定了其在复合物中包埋及不同pH介质中的释药行为.结果表明,BSA可以在非常温和条件下有效包埋于复合物中,包埋率接近100%;BSA从复合物中释放具有很高的pH响应性,释放转变在很窄的pH范围内(<0.4pH单位)完成,释放转变临界pH(pH′Φ)可由两性壳聚糖中丙烯酸取代度调控.复合物形成和蛋白质释放在对pH依赖性上存在很好的相关性.同时还发现,在中性介质中(pH7.4),复合物对BSA具有很好的缓释作用,BSA持续释放时间可达15天左右.     

Equilibrium ion exchange method: methodology at low ionic strength and copper(II) complexation by dissolved organic matter in a leaf litter extract

The equilibrium ion exchange method (EIM) is a powerful tool for the investigation of metal cation complexation by dissolved organic matter (DOM) in natural systems. Tests with different ion exchange resins demonstrated that under low ionic strength conditions (0.01 mol/kg) and in the presence of DOM, equilibration times of at least 24 h are required for experiments with Cu(II). The classical approach to the EIM was modified by using nonlinear reference adsorption isotherms in order to expand the method to a broader range of experimental conditions. For Cu(II) at low ionic strength (0.01 mol/kg), the reference isotherms between pH 4 and 6 were identical and were mathematically modeled in terms of Langmuir adsorption parameters. The EIM using nonlinear reference isotherms was validated between pH 4 and 6 by the correct determination of the stability constants for the complexes CuOxalate and Cu(Oxalate)(2). Then the method was used to quantitatively characterize the Cu(II) complexation behavior of DOM in an aqueous chestnut leaf litter extract between pH 4 and 6. In contrast to the classical approach to the EIM, data were analyzed by using plots [Cu](bound)/[Cu](free)vs. [Cu](bound). This allowed the determination of both, conditional stability constants and metal binding capacities for two different binding site classes. The logarithmic values of the stability constants were about 8 for the strong binding sites and 5.5-6 for the weak binding sites. The total Cu(II) binding capacity increased from 0.22 mol/(kg C) at pH 4 to 2.85 mol/(kg C) at pH 6.