Spectroscopic analysis and drug-binding studies of the CNBr fragments of human serum albumin

Three large fragments (A, B and C) of human serum albumin (HSA) were produced by cyanogen bromide digestion of HSA in order to investigate the specific binding sites. The fragments were isolated by use of gel filtration, followed by high performance ion exchange chromatography. The isolated fragments were examined by use of UV/Vis, steady-state fluorescence, and circular dichroism spectroscopy. The study was extended to examine the interactions of bilirubin and two anionic drugs, warfarin and naproxen, with HSA and the three fragments. The primary bilirubin binding site on HSA molecule appeared to be located between fragment A and fragment C. The results also suggest the binding sites of the two anionic drugs to mosy likely be located in fragment C of HSA molecule.     

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.     

Characterization of thyroxine-albumin binding using high-performance affinity chromatography. I. Interactions at the warfarin and indole sites of albumin.

A high-performance affinity column containing immobilized human serum albumin (HSA) was used to study the binding of thyroxine at the warfarin and indole sites of HSA. Frontal analysis, using R-warfarin and L-tryptophan as probes for these sites, demonstrated that the immobilized HSA had binding behavior equivalent to that observed for HSA in solution. By injecting R-warfarin or L-tryptophan in the presence of excess thyroxine, it was found that thyroxine was binding directly to both types of site. The warfarin and indole sites had relatively strong binding for thyroxine, with association constants at 37 degrees C of 1.4 x 10(5) and 5.7 x 10(5) M-1, respectively. The value of delta G for these sites ranged from -7 to -8 kcal/mol and had a significant entropy component. The techniques used in this study are not limited to thyroxine-HSA interactions, but should also be valuable in examining the site-specific binding of other drugs and hormones to HSA.     

7-Alkylaminocoumarin-4-acetic acids as fluorescent probe for studies of drug-binding sites on human serum albumin

7-Alkylaminocoumarin-4-acetic acids I-IX having alkylamino groups different in alkylchain lengths were synthesized as fluorescence probes for characterization of drug-binding sites on human serum albumin (HSA). The fluorescences of I-IX were quenched or enhanced in the presence of HSA with shifts of the emission maxima to shorter wavelength. The binding constants and the number of binding sites were determined by the spectral changes of the probes I-IX bound to HSA through analysis of Scatchard's and Job's plots. The primary binding sites of the tested probes were found to be site 2 (diazepam site) on HSA from the results of competitive displacement studies. The polarity of site 2 was estimated from the relationship between the emission maximum of the probe of IV and Z-values, and was found to be comparable to that of acetonitrile. Simple attempts to estimate the site 2 region from the molecular size of the probe of VIII obtained using the Corey-Pauling-Koltun molecular model suggest that the hydrophobic cleft at site 2 is about 21-25 A in depth. The distance between the lone tryptophan residue in HSA and probes bound to site 2 was estimated to be 15-17 A using F?rster's equation on the basis of fluorescence energy transfer. The present data suggest that I-IX are useful as fluorescence probes for the characterization of site 2 on HSA.     

高效亲和色谱法测定2种中药成分与人血清白蛋白的结合率

采用高效亲和色谱技术(HPAC)对中药成分与人血清白蛋白(HSA)的相互作用进行了研究。首先采用点击化学的方法制备了表面键合有HSA蛋白的硅胶固定相并装填成亲和色谱柱,根据药物在该色谱柱上与空白硅胶柱上的保留时间差计算得到药物与蛋白的结合率。利用该方法测得模型化合物华法令与HSA的结合率与文献中采用超滤法测得的结果基本一致,表明该方法可用于测定药物与HSA的结合率。在此基础上用该方法测定了葛根素和告依春两种中药成分与HSA的相对结合率分别为10.26%和10.20%。同时用超滤的方法测定了葛根素与HSA的结合率为14.25%。结果表明,HPAC可以作为研究药物与蛋白相互作用的一种简便可行的方法,其测定结果与超滤方法一致。     

Binding of caffeic acid to human serum albumin by the retention data and frontal analysis

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high‐performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 10⁴/m . The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm . The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug–protein interaction. The proposed model also has the advantages of ligand saving and rapid operation. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a disulfiram-modified human serum albumin-based HPLC column

Summary A human serum albumin (HSA)-based HPLC column has been modified in situ by disulfiram, an alcohol-deterrent drug reported to bind cys₃₄, the only free cysteine in HSA, under physiological conditions. The reversible and covalent binding of disulfiram was found to change the binding properties of the protein, giving rise to a new selector which performed differently from the native albumin-based stationary phase. When low concentrations of disulfiram were used as mobile phase modifier, reversible binding resulted in a cooperative allosteric effect with improved selector performance. Covalent modification resulted in markedly reduced affinity for binding of the drugs to sites I or II, while still maintaining enantioselectivity. This study has enabled the monitoring of interactions of disulfiram with potentially coadministered drugs, and the preparation of a chiral selector with different drug affinity and enantioselectivity.     

Bromocresol green as a new spectrophotometric probe for serum albumins

Bromocresol green (BCG) has been employed as a new spectrophotometric probe to characterise the binding regions of human serum albumin (HSA) and bovine serum albumin (BSA). BCG binds with greater affinity onto BSA than onto HSA. Based on the abilities of ligands Naproxen and l-anilino-8-naphthalenesulphonic acid (ANS) to displace BCG from the serum albumins by competitive or non-competitive mechanism, binding regions were identified for these ligands. It has been found that both Naproxen and ANS share common binding sites with BCG in HSA with the relative ability of Naproxen > ANS on binding to HSA. In the case of BSA, ANS competes with BCG for the same binding sites, whereas Naproxen exhibits non-competitive binding. The highaffinity sites of Naproxen coincide with BCG binding sites while the low-affinity sites occur at sites distinct from the BCG binding region.     

酮洛芬与蛋白结合作用的高效液相色谱-迎头分析法研究及其与高效毛细管电泳-迎头分析法的比较

采用高效液相色谱-迎头分析法(HPLC-FA),以67 mmol/L (pH 7.4, I=0.17 mol/L) 的等渗磷酸盐缓冲液为流动相,Pinkerton GFF　Ⅱ-S5-80内表面反相柱(150 mm×4.6 mm i.d., 5 μm)为固定相,254 nm下检测,研究了酮洛芬与人血清白蛋白(HSA)的结合作用,通过非线性回归参数估算求得酮洛芬与HSA的结合参数。与高效毛细管电泳-迎头分析法(HPCE-FA)相比,HPLC-FA法具有高灵敏度的优势,但进样量较大,分析时间较长。HPLC-F     

Dehydroalanine derived from cysteine is a common post-translational modification in human serum albumin

The conversion of a cysteine residue into dehydroalanine (DHA) in proteins was previously described. This post-translational modification (PTM) can be generated artificially as a result of heat and an alkaline environment. The presence of this PTM on human serum albumin (HSA) in plasma collected from healthy volunteers and critically ill patients as well as in commercially available HSA was studied. Using liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) methods, a fragment containing DHA was identified in the trypsin digest of commercial HSA and isolated HSA from plasma. The sequence (RPC*FSALEVDETYVPK) corresponded to the expected molecular mass and fragmentation pattern of a tryptic peptide of HSA where the cysteine residue (cys487) was modified to DHA. The presence of this common PTM of HSA has potential effects on ligand binding to HSA, plasma clearance of this oxidized form of HSA, protein-protein interactions, and oxidation-reduction potential.     

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.     

A Thermodynamic Study on the Binding of Human Serum Albumin with New Synthesized Anti Cancer Pd (II) Complex

The thermodynamics of the interaction between new synthesized anti-cancer drug (2,2′-bipyridin n-butyl dithiocarbamato Pd (II), ButPd), and HSA was investigated at pH=7 by isothermal titration calorimetry. A new solvation model was used to reproduce the enthalpies of HSA interaction by ButPd within a broad range of complex concentrations. The solvation parameters attained from the new model were attributed to the structural change and biological activity of HSA. The binding parameters for the interaction of ButPd and HSA indicated that the considerable conformational changes in HSA were not observed after being bound with ButPd. It was found that HSA has three identical and cooperative binding sites for ButPd.     

Multiple and irreversible binding of cis-diamminedichloroplatinum(II) to human serum albumin and its effect on warfarin binding.

Irreversible bindings of cis-diamminedichloroplatinum(II) (cis-DDP) to human serum albumin (HSA) were investigated in a pH 7.4 buffer containing 0.1 M NaCl at various molar ratios (cis-DDP/HSA) up to 60 over a 14 d period (37 degrees C). The metal binding seemed to reach a plateau when incubated at less than 10 times excess of cis-DDP. As the molar ratio increased, the reaction rate was relatively fast within the first day, followed by a moderate increase in the metal binding. When incubated at 60 times excess of cis-DDP, the metal bound as much as 20 mol per mol of HSA in 14 d. Fluorescence quenching of the metal-bound protein suggested that the tryptophan residue was gradually exposed to a hydrophilic environment as the metal binding increased. Furthermore, cis-DDP cleaved disulfide bonds at the ratio of 1 mol of disulfide bond per 5.3 mol of the metal binding. It was therefore suggested that the metal binding also occurred at several sites other than the disulfide bond. Warfarin binding to the metal-bound protein, examined by fluorescence changes, also decreased with increasing metal binding or cleavage of the disulfide bonds. Thus, cis-DDP bound to multiple sites in addition to the lone sulfhydryl group (Cys-34), suggesting that massive conformational changes of the protein took place.     

Chromatographic study of the adsorption kinetics of albumin on monoclonal and polyclonal immunoadsorbents

Summary High-performance liquid chromatography (HPLC) has been used to study the adsorption kinetics of proteins on immunoadsorbents. The adsorption rate constant of human serum albumin (HSA) on monoclonal and polyclonal anti-HSA antibodies immobilized on a silica HPLC support was determined by saturating the column with repeated pulse injections. Studies on polyclonal immunodsorbents of different capacities enable evaluation of the contribution of transport to the binding sites. The adsorption properties of two different monoclonal anti-HSA antibodies immobilized on a chromatographic support were characterized by different approaches. The location of the epitope on the HSA molecule was determined by enzyme-linked immunoassay (ELISA) with albumin fragments. The chromatographic method was used to determine the column capacity and the adsorption rate constant of HSA on the immunoadsorbent. To compare the affinity of the antibodies for the antigen, an indirect ELISA method was used to determine the equilibrium constant of antigen-antibody association in solution Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th september, 1996.     

Zinc-human serum albumin association: testimony of two binding sites

The zinc cation (Zn²⁺) binding to human serum albumin (HSA) was studied using a non-equilibrium approach in order to prove two HSA binding sites. The effect of the bulk solvent pH and column temperature T on this binding and the corresponding thermodynamic data were also investigated. It appeared that the association process can be divided into two pH value ranges due to a predominant Zn²⁺ interaction with either HSA site I or site II. It was also demonstrated that the Zn²⁺ affinity for the site II was weakly affected by modifying the mobile phase pH whereas for the site I, the affinity constant increased strongly with increasing the pH of the bulk solvent.     

光谱法研究哈巴俄苷与人血清白蛋白的结合反应

在不同温度及模拟血液pH值条件下,采用荧光光谱法和紫外-可见吸收光谱法研究了哈巴俄苷(Harpagoside, HAR)与人血清白蛋白(Human serum albumin, HSA)的结合反应.结果表明,HAR有规律地使HSA内源荧光猝灭,猝灭常数随温度升高而降低,其猝灭机制为两者形成复合物而引起的的静态猝灭;不同条件下两者结合常数KA均大于105 L/mol,结合位点数n≈1.由Van′t Hoff方程计算获得了不同条件下HAR与HSA相互作用的热力学参数,由ΔG、ΔH和ΔS均小于0可知,两者结合的主要作用力是氢键和范德华力,且两者结合是吉布斯自由能降低的自发过程.根据F(o)rster非辐射转移理论计,计算了不同条件下HAR与HSA的结合距离r在4.01~4.28 nm范围内,表明两者结合过程发生了非辐射能量转移.同步荧光光谱表征结果表明,HAR使HSA的色氨酸和酪氨酸残基所处的微环境极性增强,疏水性减弱,导致HSA构象发生了一定程度的改变.     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法,表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息.同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境;二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光,使其构象发生变化.荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息,揭示KA的存在使HSA的流动性和微粘度发生变化.定量求得不同温度下(298、308和318 K)考拉维酸与HSA作用的键合参数和热力学参数.分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内,并与赖氨酸Lys195和天冬氨酸Asp451形成三个氢键,与HSA的键合模式主要是疏水作用;位点竞争实验证明考拉维酸在HSA亚结构域的位点II位发生作用.另外,获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制.结果表明,HSA对考拉维酸有较强的结合能力,提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法, 表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息. 同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境; 二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光, 使其构象发生变化. 荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息, 揭示KA的存在使HSA的流动性和微粘度发生变化. 定量求得不同温度下(298、308 和318 K)考拉维酸与HSA作用的键合参数和热力学参数. 分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内, 并与赖氨酸Lys195 和天冬氨酸Asp451 形成三个氢键, 与HSA的键合模式主要是疏水作用; 位点竞争实验证明考拉维酸在HSA亚结构域的位点Ⅱ位发生作用. 另外, 获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制. 结果表明, HSA对考拉维酸有较强的结合能力, 提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

Selectivity of monolithic supports under overloading conditions and their use for separation of human plasma and isolation of low abundance proteins

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These two proteins frequently interfere with detection, determination and purification of low abundance proteins that can be potential biomarkers and biomarker candidates for various diseases. Some low abundance plasma proteins such as clotting factors and inhibitors are also important therapeutic agents. In this paper, the characterization of ion-exchange monolithic supports under overloading conditions was performed by use of sample displacement chromatography (SDC). If these supports were used for separation of human plasma, the composition of bound and eluted proteins in both anion- and cation-exchange mode is dependent on column loading. Under overloading conditions, the weakly bound proteins such as HSA in anion-exchange and IgG in cation-exchange mode are displaced by stronger binding proteins, and this phenomenon was not dependent on column size. Consequently, small monolithic columns with a column volume of 100 and 200 μL are ideal supports for high-throughput screening in order to develop new methods for separation of complex mixtures, and for sample preparation in proteomic technology.