金属元素组与血清白蛋白的竞争结合反应性能研究

利用亲和毛细管电泳(Affinity capillary electrophoresis,ACE)法研究金属元素组和血清白蛋白(Bovine serum albumin,BSA)的竞争结合反应性能。基于位点结合模型,构建双金属组[Zn2+,Cu2+]与血清白蛋白结合反应模型,建立多元金属组与生物大分子竞争结合的理论方程,测定结合参数并解析动力学机制。结果表明,金属元素组[Zn2+,Cu2+]与BSA发生竞争结合反应形成配合物Zn2+-BSA和Cu2+-BSA。依据有效淌度变化,通过建立的理论方程非线性拟合竞争结合反应的平均表观结合常数KZn2+-BSA=4.01×104L·mol-1、KCu2+-BSA=7.75×104L·mol-1。结合反应均为快平衡反应,Cu2+对Zn2+离子的结合作用有明显拮抗作用。分析ACE谱显示配合物的峰高与配体结合能力大小、配合物稳定性之间存在量效关系。     

Fe离子不同形态诱导血清蛋白质分子别构效应的比较分析研究

通过紫外差谱方法研究金属Fe离子不同形态与不同类别血清白蛋白分子的别构效应,并比较分析分子作用机理.考察氧介导条件对Fe离子不同形态分别与人血清白蛋白(Human serum albumin,HSA)、牛血清白蛋白(Bovine serum albumin,BSA)别构效应的影响,建立定量模型方程.结果表明,Fe离子不同形态与血清白蛋白结合反应体系中存在Fe(Ⅱ)-HSA/BSA～Fe(Ⅲ)-HSA/BSA的动态平衡,Fe(Ⅱ)-HSA/BSA～Fe(Ⅲ)-HSA/BSA电子转移效应是别构效应的关键影响因素,导致Fe离子不同形态与血清白蛋白结合反应的别构效应迥异,呈现形态显著性差异.氧介导及无氧条件下的Fe(Ⅱ)-HSA/BSA～Fe(Ⅲ)-HSA/BSA电子转移效应机理不同.无氧条件下,Fe(Ⅲ)与血清白蛋白的相互作用遵循动力学一级反应规律,计算得到结合反应体系的速率常数k及自由能变ΔG≠.     

光谱法研究黄曲霉毒素B1与人血清白蛋白的结合反应

黄曲霉毒素B1(AFB1)是目前发现的致癌能力最强的真菌毒素,严重危害人畜健康.人血清白蛋白(Human serum albumin,HSA)在结合、运输内源性和外源性等小分子物质方面具有重要的生理功能.研究AFB1与HSA的相互作用机理和作用过程,在分子毒理学上具有重要意义.本研究模拟在人体血液pH条件(pH7.4,离子强度0.1 mol/L),通过荧光淬灭、3D荧光法和圆二色谱(Circular dichroism,CD)等光谱方法研究AFB1与人血清蛋白的相互作用.结果表明,AFB1与HSA的内源荧光淬灭属于静态淬灭,AFB1-HSA在298,303,308和313 K4个温度条件下,结合常数均为104数量级,结合位点都约为l.根据Van't Hoff方程,AFB1-HSA体系是熵增焓减的自发过程,分子间主要作用力为疏水作用和氢键.基于F(o)rster's能量转移,得知AFB1与HSA结合距离为3.31 nm.竞争结合实验表明,AFB1结合在HSA的siteI位点上,靠近色氨酸Trp-214.通过3D荧光分析,AFB1的结合作用导致了HSA氨基酸残基微环境和二级构象发生变化.圆二色谱的分析结果表明,二者的结合使得HSA的α-螺旋含量增加.     

La(Ⅲ)与HSA或BSA的结合平衡研究

用平衡透析法详细研究了pH=6.3条件下La(Ⅲ)与HSA或BSA的结合平衡.Scatchard图分析表明,La(Ⅲ)在HSA中有2个强结合部位和8个弱结合部位;在BSA中有2个强结合部位和6个弱结合部位.从La(Ⅲ)与Cu(Ⅱ),Zn(Ⅱ)和Cd(Ⅱ)等的竞争结合HSA或BSA的结果推测:La(Ⅲ)在HSA或BSA中的一个强结合部位的配位原子可能全部是氧原子.通过非线性最小二乘法拟合Bjerrum方程,首次报道了La(Ⅲ)-HSA和La(Ⅲ)-BSA体系的逐级稳定常数值,其K₁的数量级为10⁴.Hill系数及自由能偶合分析表明La(Ⅲ)与HSA或BSA的结合均产生一定的负协同效应.     

利福布汀与人血清白蛋白相互作用的光谱研究

用荧光光谱法和圆二色谱法研究了利福布汀(RB)与人血清白蛋白(HSA)的相互作用. 结果表明, RB与HSA之间的相互作用主要是疏水作用, 作用机制是静态猝灭与动态猝灭的结合. 其结合常数(Ka)在106数量级, 说明RB和HSA有很强的结合. 此外, 探讨了金属离子(Cu2+, Zn2+, Mg2+ 和Ca2+)对RB与HSA结合常数的影响. 同步荧光光谱和圆二色谱数据表明, RB可导致HSA的构象改变.     

烟酸修饰尾式卟啉的合成及其与人血清白蛋白的相互作用

合成了烟酸分子修饰的自由卟啉o-(niacin)C2O-T(3p-OCH3)PP、p-(niacin)C2O-T(3p-OCH3)PP及锌配合物o-(niacin)C2O-T(3p-OCH3)PPZn、p-(niacin)C2O-T(3p-OCH3)PPZn.经元素分析、紫外-可见光谱、核磁共振氢谱(1HNMR)、红外(IR)光谱等对结构进行了表征,并通过量子化学方法计算了锌卟啉的最低能量构型.实验结果表明:o-(niacin)C2O-T(3p-OCH3)PPZn中侧链烟酸基团处于卟啉环上方,烟酸基团中N原子与卟啉环中Zn2+存在着Zn―N间的分子内配位作用,而p-(niacin)C2O-T(3p-OCH3)PPZn中侧链烟酸基团处于卟啉环较远的位置,一个锌卟啉的中心Zn2+与另一个锌卟啉烟酸中N原子之间存在着Zn―N间的分子间配位作用.同时,为模拟金属卟啉的生物功能,采用荧光光谱滴定法测定了金属锌卟啉与人血清白蛋白相互作用的光谱性质.荧光光谱实验结果显示:金属锌卟啉与人血清白蛋白(HSA)之间发生了较强的静态荧光猝灭作用,反应机理是以氢键或van der Waals力结合反应.按照Stern-Volmer方程、Lineweaver-Burk双倒数方程分析和处理实验数据,得到了反应的猝灭常数、结合常数和热力学参数等.     

La（III）与HSA或BSA的结合平衡研究

用平衡透析法详细研究了pH=6.3条件十La(III)与HSA或BSA的结合平衡,Scatchard图分析表明,La(III)在HSA中有2个强结合部位和8个弱结合部位,在BSA中有2个强结合部位和6个弱结合部位,从La(III)与Cu(II),Zn(II)和Cd(II)等的竞争结合HSA或BSA的结果推测,La(III)在HSA或BSA中的一个强结合部位的配位原子可能全部是氧原子,通过非线性最小二乘法拟合Bjerum方程,首次报道了La(III)-HSA和La(III)-BSA体系的逐级稳定常数值,其K1的数量极为10^4,Hill系数及自由能偶合分析表明La(II)与HSA或BSA的结合产生一定的负协同效应。     

Cd(II)与HSA或BSA的结合平衡研究

用平衡透析法详细研究了生理pH(7.43)条件下Cd(II)与HSA或BSA的结合平衡。通过非线性最小二乘法拟合Bjerrum方程,首次报道了Cd(II)-HSA和Cd(II)-BSA体系的逐级稳定常数值,其K~1-K~3的数量级均为10^4;Hill系数和自由能偶合定量分析表明Cd(II)与HSA或BSA的结合均产生在类似体系中少见的强的正协同效应,且Cd(II)与HSA结合产生的正协同效应大于BSA;Scatchard图分析表明,Cd(II)在HSA和BSA中均有3个强结合部位。通过Cd(II)与Cu(II),Zn(II)或Ca(II)等竞争结合HSA或BSA的结果,进一步讨论了Cd(II)在HSA或BSA中强结合部位的可能位置和(或)配体。     

微透析取样法研究金属离子与蛋白质的竞争结合作用

采用微透析结合高效液相色谱法建立了一种研究金属离子与蛋白质竞争结合作用的方法. 采用Cu²⁺, Ni²⁺, Cd²⁺, Zn²⁺与人血清蛋白(human serum albumin, HSA)为模型化合物, 通过各种离子之间的竞争结合的研究表明: 在HSA上Cu²⁺和Ni²⁺有共同的第一结合位点, Cd²⁺ 和Zn2+有共同的第一结合位点, 它们在HSA上有一多离子的共同结合位点. 本方法具有通用、快速、方便等特点, 可用于无理想谱学探针的蛋白质-金属离子相互作用体系的研究.     

Cd（Ⅱ）与HSA或BSA的结合平衡研究

用平衡透析法详细研究了生理pH(7.43)条件下Cd(Ⅱ)与HSA或BSA的结合平衡.通过非线性最小二乘法拟合Bjerrum方程,首次报道了Cd(Ⅱ)-HSA和Cd(Ⅱ)-BSA体系的逐级稳定常数值,其K_1～K_3的数量级均为10~4;Hill系数和自由能偶合定量分析表明Cd(Ⅱ)与HSA或BSA的结合均产生在类似体系中少见的强的正协同效应,且Cd(Ⅱ)与HSA结合产生的正协同效应大于BSA;Scatchard图分析表明,Cd(Ⅱ)在HSA和BSA中均有3个强结合部位.通过Cd(Ⅱ)与Cu(Ⅱ),Zn(Ⅱ)或Ca(Ⅱ)等竞争结合HSA或BSA的结果,进一步讨论了Cd(Ⅱ)在HSA或BSA中强结合部位的可能位置和(或)配体.     

Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/R_f), which provided information about the binding strength and the overall charge of the protein‐ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments.     

Affinity capillary electrophoresis and density functional theory employed for the characterization of hexaarylbenzene-based receptor complexation with alkali metal ions

In this study, affinity capillary electrophoresis (ACE) and quantum mechanical density functional theory (DFT) calculations were combined to investigate non-covalent binding interactions between the hexaarylbenzene-based receptor (R) and alkali metal ions, Rb(+) and Cs(+) , in methanol. The apparent binding (stability) constants (K(b) ) of the complexes of receptor R with alkali metal ions in the methanolic medium were determined by ACE from the dependence of effective electrophoretic mobility of the receptor R on the concentration of Rb(+) and Cs(+) ions in the BGE using a non-linear regression analysis. The receptor R formed relatively strong complexes both with rubidium (log K(b) =4.04±0.21) and cesium ions (log K(b) =3.72±0.22). The structural characteristics of the above alkali metal ion complexes with the receptor R were described by ab initio density functional theory calculations. These calculations have shown that the studied cations bind to the receptor R because they synergistically interact with the polar ethereal fence and with the central benzene ring via cation-π interaction.     

Thermodynamics of porphyrin binding to human serum albumin using affinity capillary electrophoresis

Summary The interaction thermodynamics of heptacarboxylporphyrin (HCP) and protoporhyrin (PP) with human serum albumin (HSA) was studied by affinity capillary electrophoresis (ACE) over the temperature range of 25–50°C, where HCP and PP bound to HSAvia 1:1 molecular association. The binding equilibrium constants (pH 7.4, phosphate buffer) for the binding of HCP with HSA were found to decrease with an increase in temperature, whereas the binding constants of the PP/HSA system appeared to be independent of temperature changes over the range studied. The van’t Hoff relationship (25–50°C) was found to be linear for the interaction of either HCP or PP with HSA. However, the interaction thermodynamics for both of these porphyrins with HSA were found to be quite different. In particular, the interaction of HCP (a hydrophilic porphyrin) with HSA appeared to be based on an enthalpy-driven process, whereas the binding between PP (a hydrophobic porphyrin) and HSA driven by a favorable change in entropy. The ability of using ACE to evaluate the interaction thermodynamics of serum proteins (e.g., HSA) with ligands (e.g., porphyrins and related compounds) should aid in the development of new and more effective photosensitizers in the photodynamic therapy of cancer.     

Study of the interactions between fluoroquinolones and human serum albumin by affinity capillary electrophoresis and fluorescence method

The interactions between fluoroquinolones and human serum albumin (HSA) were investigated by affinity capillary electrophoresis (ACE) and fluorescence quenching technique. Based on the efficient separation of several fluoroquinolones using a simple phosphate buffer, the binding constants of fluoroquinolones with HSA were determined simultaneously during one set of electrophoresis by ACE method. The thermodynamic parameters were obtained from data at different temperatures, and the negative ΔH and ΔS values showed that both hydrogen bonds and van der Waals interaction played major roles in the binding of fluoroquinolones to HSA. The interactions were also studied by fluorescence quenching technique. The results of fluorescence titration revealed that fluoroquinolones had the strong ability to quenching the intrinsic fluorescence of HSA through the static quenching procedure. The binding site number n, apparent binding constant K_b and the Stern-Volmer quenching constant K_sv were determined. The thermodynamic parameters were also studied by fluorescence method, and the results were consonant with that of ACE.     

Protonated diamines as anion-binding agents and their utility in capillary electrophoresis separations

Capillary zone electrophoresis is a proven method for separating small ions because of the inherent charge and differences in mobility of these analytes. Despite its resolving power, CZE can be insufficient for separating ions with similar mobilities. One remedy is to modify mobilities via the addition of background electrolyte complexation agents. However, this approach is not straightforward for inorganic anions, which lack complexation options. To address this shortfall, the diprotonated diamine moiety was investigated for complexation of dianions. Dicationic diamines significantly complexed dianions, and this interaction was not purely electrostatic in nature because affinities varied with dianion identity. Aqueous association constants were measured with affinity capillary electrophoresis (ACE) and found to be similar in magnitude but different in selectivity to those of dianions with magnesium ion. Binding was also investigated for zwitterionic buffers containing the protonated diamine moiety. Zwitterions exhibited binding constants as high as 18 M^?1 (30‐mM ionic strength). This work discusses the observed binding constants and their potential usefulness in CZE separations of inorganic anions. Also covered are improvements to ACE methodology and an evaluation of some of the assumptions employed.     

Optimization of affinity capillary electrophoresis for routine investigations of protein–metal ion interactions

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra‐ and inter‐instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra‐instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter‐instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein–metal ion interactions (relative standard deviation of 0.16–0.89%, 15 series, 12 runs for each).     

Preparation of an iminodiacetic acid-modified capillary and its performance in capillary liquid chromatography and immobilized metal chelate affinity capillary electrophoresis

We prepared iminodiacetic acid (IDA)-modified and Cu(II)-IDA-modified capillaries through polymerization of N-(vinylbenzylimino) diacetic acid. The fundamental performance of these capillaries was examined in capillary liquid chromatography (LC) and immobilized metal chelate affinity capillary electrophoresis (IMACE). Copper(II), cobalt(II), and hematin were detected at different retention times by means of capillary LC with a chemiluminescence detector, during which the IDA-modified capillary was used. The difference in the retention times was attributed to the difference in the interaction between metal ions or complex and IDA moieties on the inner wall of the capillary. In addition, human serum albumin (HSA) and human serum gamma-globulin (HgammaG) were separated and detected using IMACE with an absorption detector, during which the Cu(II)-IDA-modified capillary was used. The separation of HSA and HgammaG was achieved through the interaction between proteins and Cu(II) chelate moieties on the inner wall of this capillary.     

Determination of the binding parameters for antithrombin-heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.     

Binding studies of porphyrins to human serum albumin using affinity capillary electrophoresis

The present work demonstrates that affinity capillary electrophoresis (ACE) can be employed as a valuable and powerful tool for studying the interactions between porphyrins and proteins in biological and biomedical research, such as the development of porphyrins and related compounds as efficient and selective photosensitizers in the photodynamic therapy of cancers. Binding constants of human serum albumin (HSA) to four biological porphyrins (uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, protoporphyrin IX), which possess a wide range of hydrophobicity, were estimated by ACE. Based on 1:1 molecular association between these individual porphyrins and HSA, the change of the electrophoretic mobility of HSA as a function of porphyrin concentration in the run buffer was measured and the binding constants were calculated from the slope of the Scatchard plots. The binding constant values were found to be 8.80 +/- 0.51 x 10(4) M(-1), 2.39 +/- 0.16 x 10(5) M(-1), 1.61 +/- 0.11 x 10(6) M(-1), and 9.34 +/- 0.30 x 10(6) M(-1) for uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, and protoporphyrin IX, respectively, and most of these results are in good agreement with those reported in the literature using conventional methods for binding measurements. Additionally, experimental binding constant data obtained using ACE was found to exhibit very good correlation with theoretical hydrophobicity values calculated using the Rekker's hydrophobic fragmental constant method, thus further supporting the hypothesis that the hydrophobicity of the porphyrin side chains play an important role in governing the hydrophobic interaction of porphyrins with serum proteins such as HSA.     

Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S‐omeprazole, S‐OME) and its R‐enantiomer (R‐omeprazole, R‐OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10³ M⁻¹ and 5.36 × 10³ M⁻¹, respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs.