Characterization of the interaction between 8-bromoadenosine with human serum albumin and its analytical application

This study was designed to examine the interaction of 8-bromoadenosine with human serum albumin (HSA) by fluorescence spectroscopy in combination with molecular modeling under simulative physiological conditions. The results of fluorescence measurements indicate that 8-bromoadenosine has a strong ability to quench the intrinsic fluorescence of HSA through static quenching procedure. The binding constants (K) at different temperatures and thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated according to the fluorescence data. The results showed that the hydrophobic force played the major role in the binding of 8-bromoadenosine to HSA. The fluorescence experimental results were in agreement with the results obtained by molecular modeling study. The effects of some normal positive and negative ions on the binding constants were also discussed. Moreover, the synchronous fluorescence technique was used to characterize the interaction of 8-bromoadenosine to HSA and successfully applied to determine the total proteins in human serum, urine and saliva samples at room temperature under the optimum conditions with a wide linear range and satisfactory results.     

Evaluation of an immunoaffinity extraction column for enrichment of adducts between human serum albumin and hexahydrophthalic anhydride in plasma

An immunoaffinity extraction (IAE) column was prepared for extraction of adducts between human serum albumin (HSA) and hexahydrophthalic anhydride (HHPA). HHPA is a strong sensitizer inducing immunoglobulin E antibodies in vivo. Polyclonal antibodies from a rabbit immunized with keyhole limpet hemocyananin-HHPA conjugate were purified using a Protein A Sepharose gel. To obtain antibodies with optimal affinity towards HHPA-protein adducts, HHPA-specific antibodies were selected using an N-hydroxysuccinimide-Sepharose column coupled with albumin-HHPA conjugate. Antibodies eluted from this column at pH 2.2 were selected to prepare the IAE column. The column was evaluated using 2 mL plasma spiked with HSA-HHPA conjugate. The column was eluted with glycine buffer at pH 2.0. The conjugates in the eluate were hydrolyzed to the corresponding HHP acid and quantified by mass spectrometry. The average recovery of HHPA adducts in 11 experiments was 68% with a coefficient of variation (CV) of 7%. The column's capacity to bind protein-HHPA adducts was found to be linear in the range of 0.15-1.2 nmol conjugate. The evaluation showed that the IAE column had adequate affinity towards the HHPA adducts and that the adducts could be extracted with good recovery and precision from a large volume of plasma.     

白藜芦醇类似物与人血清白蛋白的相互作用

采用荧光光谱、三维荧光光谱、紫外吸收光谱研究白藜芦醇类似物(Z)-2-(3,4-二甲氧苯基)-3-(4-二甲氨基苯基)丙烯腈(HCQ)与人血清白蛋白(HSA)的相互作用,探讨其作为抗肿瘤药物的可能。结果发现HCQ与HSA形成了基态配合物,HCQ主要结合于HSA的位点Ⅰ,与位点Ⅱ也有微量的结合,反应为自发的放热反应,其ΔH、ΔS、ΔG均小于零,二者之间的结合力为氢键或者范德华力,结合常数为104～105数量级。HCQ与HSA的结合使HSA构象发生变化,Trp-214所处的环境疏水性增加,使得其内源性荧光显著降低。说明合成的白藜芦醇类似物能够与人血清白蛋白结合。     

氟罗沙星与人血清白蛋白相互作用研究

以光谱技术和微量热技术相结合的方法研究水溶液中氟罗沙星分子及铁存在时与人血清白蛋白之间结合作用的机制,用荧光淬灭法测得两反应的结合常数分别为K=1.19×l05L·mol-1和1.0565×105L·mol-1,结合位点数0.97和0.835.依据Fōrster非辐射能量转移机制,得到氟罗沙星与人血清白蛋白间的结合距离(r=3.23nm).用同步荧光技术考察氟罗沙星对人血清白蛋白构象的影响.微量热法测得氟罗沙星与人血清白蛋白反应的ΔH≈0,ΔS＞0,氟罗沙星分子在铁离子存在时与人血清白蛋白反应的ΔH＜0,并且ΔS＞0表明它们的作用都主要为静电力.由于焓熵互补的作用,两反应的自由能没有发生大的变化.     

Characterization of binding sites for sulfadimethoxine and its major metabolite, N4-acetylsulfadimethoxine, on human and rabbit serum albumin

In order to gain an understanding of protein binding of sulfadimethoxine (SDM) and its major metabolite, N4-acetylsulfadimethoxine (N4-AcSDM), the binding of SDM and N4-AcSDM to human and rabbit serum albumin (HSA and RSA) was investigated using circular dichroism (CD), fluorescence and dialysis techniques. The CD spectral characteristics of the compounds bound to the albumins suggested that the drug-binding sites on the HSA and RSA had somewhat different asymmetries. The binding constants for SDM-HSA and -RSA interaction were smaller than those for N4-AcSDM. Two specific drug-binding sites were found on RSA, similarly to HSA, from the results of competitive displacement using fluorescence probes. Moreover, SDM and N4-AcSDM were found to share the same first binding site on the albumins. It can be presumed from the displacement data with a series of p-aminobenzoates that the characteristics of the binding sites (such as depth and width of the hydrophobic cleft) for SDM and N4-AcSDM on RSA may be almost the same, but the characteristics of these drug-binding sites on HSA may be somewhat different.     

Amyloid fibril formation and other aggregate species formed by human serum albumin association

Under in vitro solution conditions where the native state is destabilized, many proteins present an abnormal structure and metabolism associated with a strong tendency to self-aggregation into a polymeric amyloid fibril structure, suggesting that this ability is a generic feature of the polypeptide chains. Such structures play a key role in different pathogenesis of neurodegenerative diseases such as Alzheimer, Parkinson, or Creutzfeldt-Jakob. Here, we report the formation of amyloid fibrils in the plasma protein human serum albumin under different in vitro conditions monitored using a combination of spectrophotometric and microscopic techniques. Amyloid fibril formation, therefore, is also allowed in a protein with a high degree of structural complexity. We also infer from experimental data the existence of other protein aggregated species than fibrils, some of which seem to be formed by a structural rearrangement of the proper fibrils.     

Characterization of interaction between doxycycline and human serum albumin by capillary electrophoresis‐frontal analysis

The binding of doxycycline to HSA under simulated physiological conditions (pH 7.4, 67 mM phosphate, I=0.17, drug concentration 100 μM, HSA concentration up to 475 μM, 36.5°C) was studied by CE‐frontal analysis. The number of primary binding sites, binding constant and physiological protein‐binding percentage were 1.9, 1.51×10³ M^?1 and 59.80%, respectively. In addition, the thermodynamic parameters including enthalpy change (ΔH), entropy change (ΔS) and free energy change (ΔG) of the reaction were obtained in order to characterize the acting forces between doxycycline and HSA. Furthermore, to better understand the nature of doxycycline–HSA binding and to get information about potential interaction with other drugs, displacement experiments were performed. The results showed that doxycycline binds at site II of HSA.     

Characterization of the interaction between a new merocyanine dye and bovine serum albumin

A new merocyanine dye was synthesized, and its acidity constant was determined by spectrophotometric and chemometrics methods. The interactions of the new cyanine dye with bovine serum albumin (BSA) have been studied by fluorescence and UV absorption spectroscopy at pH 7.40. A visual color change from red to blue was observed by addition of BSA to aqueous solution of the dye. The quenching constants and binding parameters (binding constants and number of binding sites) were determined at different temperatures. The calculated thermodynamic parameters confirmed that the binding reaction is mainly entropy-driven, whereas electrostatic interaction plays major role in the reaction. The displacement experiment confirmed binding of the dye to the subdomain IIA (site 1) of albumin. Moreover, synchronous fluorescence spectroscopy studies revealed the dye induces some local conformational change in BSA. The binding distance, r, between donor (serum albumin) and acceptor (dye) was obtained according to Förster’s theory.     

布南色林与人血清白蛋白相互作用的光谱学研究

采用荧光光谱研究了模拟生理务件下抗精神病药布南色林与人血清白蛋白的相互作用,结果表明,布南色林对人血清白蛋白的内源性荧光具有猝灭作用且猝灭方式为静态猝灭.布南色林与人血清白蛋白形成了1∶1的复合物,结合常数K=1.80×104L/mol,且金属离子对结合反应具有较显著的影响.根据不同温度下的热力学函数确定了布南色林与人...     

Spectroscopic investigation of interaction between bionanserin and human serum albumin

采用荧光光谱研究了模拟生理务件下抗精神病药布南色林与人血清白蛋白的相互作用,结果表明,布南色林对人血清白蛋白的内源性荧光具有猝灭作用且猝灭方式为静态猝灭.布南色林与人血清白蛋白形成了1∶1的复合物,结合常数K=1.80×104L/mol,且金属离子对结合反应具有较显著的影响.根据不同温度下的热力学函数确定了布南色林与人血清白蛋白的相互作用力类型以氢键和范德华力为主.同步荧光光谱和傅立叶变换红外光谱表明布南色林对人血清白蛋白二级结构的含量产生影响,α-螺旋和β-折叠的含量降低,β-转角和无卷曲规则的含量明显升高.     

水杨酸与人血清白蛋白的相互作用研究

采用荧光光谱法,紫外光谱法及圆二色谱法研究了具抗凝血作用的水杨酸与人血清白蛋白(HSA)的相互作用.结果表明,水杨酸对人血清白蛋白荧光产生猝灭现象,猝灭方式为静态猝灭.通过同步荧光法和圆二色谱法发现水杨酸的存在明显改变人血清白蛋白的构象.     

用光谱法研究奥硝唑与人血清白蛋白的相互作用

采用荧光光谱法和紫外可见吸收光谱法研究了奥硝唑与人血清白蛋白之间的相互作用;求得了二者在不同温度下的结合常数KA和结合位点数n,以及对应温度下结合反应的热力学参数,同时采用同步荧光分析技术探讨了蛋白质与药物结合时构象的变化.结果表明,在生理条件下奥硝唑对人血清白蛋白的荧光猝灭主要为静态猝灭;奥硝唑与人血清白蛋白主要靠静电作用力结合.     

Binding of glycyrrhizin to human serum and human serum albumin

The binding of glycyrrhizin (GLZ) to human serum and human serum albumin (HSA) was examined by an ultrafiltration technique. Specific and nonspecific bindings were observed in both human serum and HSA. The association constants (K) for the specific bindings were very similar: 1.31 x 10(5) M-1 in human serum and 3.87 x 10(5) M-1 in HSA. The number of binding sites (n) and the linear binding coefficient (phi) in HSA were 1.95 and 3.09 x 10(3) M-1, respectively. When the human serum protein concentration was assumed to be 4.2% (equal to the measured serum albumin concentration), n in human serum was 3.09, which is similar to the n value in HSA, and phi in human serum was 0.71 x 10(3) M-1, which is reasonably close to that for HSA. The binding pattern of GLZ with human serum protein on Sephadex G-200 column chromatography showed that GLZ binds to only the albumin fraction. It was concluded that the GLZ-binding sites in human serum exist mainly on albumin and GLZ binds to specific and nonspecific binding sites at lower and higher concentrations than approximately 2 mM, respectively.     

Molecular interaction and energy transfer between human serum albumin and polyoxometalates

As a step toward the elucidation of the mechanistic pathways governing the known bioactivity of polyoxometalates (POMs), two representative molecules of this class of chemicals, the wheel-shaped [NaP(5)W(30)O(110)]14- (P(5)W(30)) and the Keggin-type anion [H(2)W(12)O(40)]6- (H(2)W(12)), are shown, by two independent techniques, to interact with the fatty-acid-free human serum albumin (HSA). The excited-state lifetime of the single tryptophan molecule of this protein is dramatically decreased by the binding. The quenching mechanism is found to constitute the first example of energy transfer between HSA and POMs. Such molecular recognition is believed to be a key step for subsequent evolution of the systems. Circular dichroism (CD) was used to assess the structural effects of POM binding on HSA and to confirm the interaction revealed by fluorescence studies. CD experiments showed that the two POMs have different effects on the secondary structure of the protein. Binding P(5)W(30) partially unfolds the protein whereas H(2)W(12) has no remarkable effect on the structure of the protein.     

Characterization of the interaction between farrerol and bovine serum albumin by fluorescence and circular dichroism

The binding of farrerol to bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence quenching spectra, synchronous fluorescence spectra, circular dichroism (CD) and the three-dimensional (3D) fluorescence spectra at pH 7.40. The results of fluorescence titration indicated that farrerol could quench the intrinsic fluorescence of BSA in a static quenching way. The cause of showing upward curvy patterns in Stern-Volmer plots was analyzed. The binding sites number n and binding constant K using fluorescence quenching equation at 310 K were calculated. The binding distance and the energy transfer efficiency between farrerol and BSA were also obtained according to the theory of F?rster's non-radiation energy transfer. The effect of some metal ions on the binding constant of farrerol with BSA was also studied. The effect of farrerol on the conformation of BSA was analyzed using CD, synchronous fluorescence spectra and three-dimensional (3D) fluorescence spectra under experimental conditions. Furthermore, the fluorescence displacement experiments indicated that farrerol could bind to the site I of BSA.     

Spectroscopic investigation of the interaction between human serum albumin and three organic acids

The interactions of human serum albumin (HSA) with sinapic acid (SA), gallic acid (GA) and shikimic acid (SI) were investigated by fluorescence and Fourier transformed infrared spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of GA at the molar ratio of drug to HSA ranging from 0.1 to 30, and their binding constant (K(A)) is 1.1x10(4) M(-1). While one HSA molecule combined with one or two molecule of SA at the molar ratio of drug to HSA ranging from 0.1 to 4.26 or 4.26 to 30, and their binding affinities (K(A)) are 1.92x10(3) M(-1) and 6.87x10(8) M(-1), respectively. There is no specific interaction between HSA and SI. Combining the curve-fitting results of infrared amide I and amide III bands, the alterations of protein secondary structures induced by drugs were estimated. The drug-protein combination brought gradual reductions of the protein alpha-helix structure with increasing the concentrations of SA and GA, but SI did not change the protein secondary structure. From the fluorescence and FT-IR results, the binding mode was discussed in relation to the structures of the organic acids.     

Characterization of interaction kinetics between chiral solutes and human serum albumin by using high-performance affinity chromatography and peak profiling

Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2-9.6 s(-1) for the two enantiomers of m-HPPH and 3.2-4.1 s(-1) for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions.     

Monolayers of human serum albumin

Summary The influence of different factors on the spreading of human serum albumin films is studied; factors such as the ionic strength of the spreading solution, nature and concentration of the alcohol used as spreading agent, initial spreading area of the subsolution to which the protein solution was applied and the method for the spreading (direct orTrurnit). The results obtained show that the ideal spreading solution is the buffer ph=5.1,=0.01, containing 0.5% amyl alcohol (v:v). TheTrurnit's method of spreading proteins showed significant advantage over the direct deposit of drops of the protein solutions.