Mode of Interaction of Loop Diuretics with Human Serum Albumin and Characterization of Binding Site

Purpose. The purpose of this study was to investigate the binding mechanism of loop diuretics with HSA and to characterize the binding site on HSA. Methods. Quantitative analysis of potential interaction between ligands bound to HSA was performed by equilibrium dialysis and data for binding of the two ligands to HSA were analyzed on the basis of a theoretical model of simultaneous binding of two ligands. Results. The binding of loop diuretics is dependent upon the N-B transition, conformational change of albumin. Furthermore, from the results of binding of the drugs to modified HSA, the lysine residue seems to be involved in the binding of loop diuretics to HSA. Conclusions. Analysis using models describing independent, competitive, cooperative and anti-cooperative binding led to the conclusion that loop diuretics bind to site I, particularly to the warfarin region on HSA.     

Interaction of Citrinin with Human Serum Albumin

Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow’s Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions.     

Covalent Binding Between Bucillamine Derivatives and Human Serum Albumin

Purpose. To clarify the mechanism of covalent binding between human serum albumin (HSA) and drugs containing thiol groups, we studied the interactions between HSA and bucillamine (BA) and its derivatives. Methods. To determine the concentration of HSA-drug conjugate, we used columns of N-methylpyridium polymer cross-linked with ethylene glycol dimethacrylate (4VP-Me), and analyzed the reaction between HSA and B A derivatives kinetically. Following pseudo first-order reaction kinetics, the rate constants of reduction of non-mercaptoalbumin (HNA) to mercaptoalbumin (HMA) (k_a) and formation of HSA-drug conjugate (k_c) were determined. Results. Formation of HSA-drug conjugate was observed only for drugs containing one thiol group. In compound IV, the plots of k_a and k_c against pH were found to be linear. The HSA-drug conjugate was affected by various factors such as pK_a, pH, temparture and the microenviroment of Cys³⁴. The increases in k_a and k_c. against pH were mainly due to the increase in mercaptide ion concentration. Further, fatty acid affected the microenviroment of Cys³⁴, which increased HSA-drug formation. Conclusions. Cys³⁴ located in a crevice on the surface of the protein plays an important role on the formation of HSA-drug conjugate. These results may be useful for elucidating the reaction mechanisms between various proteins and thiol compounds.     

Interaction Between Two Dicarboxylate Endogenous Substances,Bilirubin and an Uremic Toxin, 3-Carboxy-4-Methyl-5-Propyl-2-Furanpropanoic Acid,on Human Serum Albumin

Purpose. Two dicarboxylate endogenous substances, bilirubin (BR) and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), have a very high affinity to human serum albumin (HSA). This study was undertaken to clarify the existence of a dicarboxylate binding site on HSA. Methods. Chemical modification, pH dependent binding and X-ray crystallographic analysis were performed to characterize these dicarboxylate binding sites. Results. It was found the binding behavior for dicarboxylates was different from typical site I ligands such as warfarin (WF) and phenyl-butazone (PB) and that electrostatic interaction was an important factor for their binding to HSA. Moreover, His residues were considered to play an important role in pH dependent binding of dicarboxylic acids but in a different manner from the site I ligands. X-ray crystallography of CMPF and BR revealed the distances between the two carboxyl groups in their chemical structures were 5.854 Å and 9.979 Å, respectively. This difference may be reflected in pH dependent binding. Using fluorescent probe displacement, we attempted to identify the binding site for monocarboxylate derivatives of CMPF and investigated the role of individual carboxyl group in the recognition of the binding site. The results suggested two carboxyl groups were important for the specific binding of CMPF to site I. Conclusions. The binding site for dicarboxylic acids is located in subdomain IIA, which includes site I, on the HSA molecule. Electrostatic interaction is an important driving force for binding to HSA.     

Interaction Mechanism Between Indoxyl Sulfate,a Typical Uremic Toxin Bound to Site II,and Ligands Bound to Site I of Human Serum Albumin

Purpose. The study was performed for clarifying the mechanism of interaction between indoxyl sulfate (IS), a typical uremic toxin bound to site II, and site I-ligands when bound to human serum albumin (HSA). The effect of the N to B transition on the interactions was also examined. Methods. Quantitative investigation of the relations between ligands bound to HSA was performed by equilibrium dialysis, and the binding data were analyzed on the basis of a theoretical model for simultaneous binding of two ligands. Results. The high-affinity binding constants for the site I-ligands warfarin (WF) and dansyl-L-asparagine (DNSA) increased with increasing pH, whereas those for the site II-ligands IS and dansylsarcosine (DNSS) were hardly affected by pH. Mutual displacement experiments showed that even though IS binds to site II it influenced binding of DNSA at the azapropazone binding area in site I. By contrast, it is unlikely that IS affects the WF binding area of site I. Furthermore, pH-profiles showed that the interaction between IS and DNSA was very sensitive to the N to B transition: competitive-like strong allosteric regulation was observed for binding of the two ligands to the N conformer (pH 6.5), whereas in the B conformation (pH 8.5) binding of these molecules was nearly independent. Conclusions. The present data provide useful information for elucidating a potential mechanism of interaction between drugs and endogenous substances including uremic toxins.     

荧光光谱法研究安非他酮与人血清白蛋白的相互作用

目的 研究安非他酮与人血清白蛋白(human serum albumin,HSA)的相互作用。方法 通过荧光光谱法研究安非他酮对HSA的荧光猝灭光谱和同步荧光光谱的影响。由Stern-Volmer方程确定安非他酮对HSA的荧光猝灭机制,双对数方程确定反应结合位点和结合常数。根据热力学方程讨论两者间主要的作用力类型。结果 荧光猝灭光谱显示,安非他酮对HSA有猝灭作用,在17 ℃和37 ℃时的猝灭速率常数分别为5.714 8×10³和3.126 1×10³ L·mol^-1·s^-1,反应前后的焓变和熵变均<0,结合点数为1。结论 安非他酮对HSA的猝灭过程为静态猝灭,二者间的结合力主要为氢键和范德华力。     

Recombinant Human Serum Albumin Dimer has High Blood Circulation Activity and Low Vascular Permeability in Comparison with Native Human Serum Albumin

Purpose Human serum albumin (HSA) is used clinically as an important plasma expander. Albumin infusion is not recommended for critically ill patients with hypovolemia, burns, or hypoalbuminemia because of the increased leakage of albumin into the extravascular spaces, thereby worsening edema. In the present study, we attempted to overcome this problem by producing a recombinant HSA (rHSA) dimer with decreased vascular permeability and an increased half-life. Methods Two molecules of rHSA were genetically fused to produce a recombinant albumin dimer molecule. The pharmacokinetics and biodistribution of the recombinant proteins were evaluated in normal rats and carrageenin-induced paw edema mouse model. Results The conformational properties of this rHSA dimer were similar to those for the native HSA (the HSA monomer), as evidenced by the Western blot and spectroscopic studies. The biological half-life and area under the plasma concentration–time curve of the rHSA dimer were approximately 1.5 times greater than those of the monomer. Dimerization has also caused a significant decrease in the total body clearance and distribution volume at the steady state of the native HSA. rHSA dimer accumulated to a lesser extent in the liver, skin, muscle, and fat, as compared with the native HSA. Up to 96 h, the vascular permeability of the rHSA dimer was less than that of the native HSA in paw edema mouse models. A prolonged plasma half-life of the rHSA dimer was also observed in the edema model rats. Conclusions rHSA dimer has a high retention rate in circulating blood and a lower vascular permeability than that of the native HSA.     

咪达唑仑与人血清白蛋白结合的荧光光谱研究

目的:研究咪达唑仑与人血清白蛋白(HSA)的结合作用。方法:分析咪达唑仑与HSA结合的荧光猝灭光谱和同步荧光光谱(激发波长和发射波长的间距(Δλ)分别为15nm和60nm);由Stern-Volmer方程确定咪达唑仑对HSA17℃和37℃时的荧光猝灭速率常数(Kq)及机制,Lineweaver-Burk双对数方程确定17℃和37℃时猝灭反应结合常数(KA)和结合位点数(n);根据反应前后的焓变ΔH和熵变ΔS的相对大小判断咪达唑仑与HSA之间的主要作用力类型。结果:荧光猝灭光谱显示咪达唑仑与HSA有荧光猝灭作用,Δλ=15nm波长的同步荧光光谱显示咪达唑仑对酪氨酸残基发生作用,未对色氨酸残基发生作用;在17℃和37℃时的Kq分别为5.0246×1011和4.757×1011L·mol-1·s-1;17℃和37℃时猝灭反应KA和n分别为2.1717×104、8.7438×103mol·L-1,1.1895、1.0769个;反应前后的ΔH和ΔS均<0。结论:咪达唑仑对人血清白蛋白的猝灭过程为静态猝灭,咪达唑仑与HSA间的作用力主要为范德华力或者氢键作用力。     

顺铂对阿霉素与人血清白蛋白结合的相互作用光谱研究

目的 研究顺铂对阿霉素与人血清白蛋白结合的相互作用。方法 采用荧光光谱法研究不同浓度顺铂对阿霉素与人血清白蛋白结合的相互作用。结果 顺铂与阿霉素对人血清白蛋白都有猝灭作用。顺铂对白蛋白的猝灭方式为动态猝灭,而阿霉素对白蛋白的猝灭方式为静态猝灭。结论 温度为17℃和37℃时,阿霉素与白蛋白的结合常数分别为2.13×104,2.76×10^4 L.mol l,2者的结合位点数为1。当加入不同浓度的顺铂后,阿霉素与白蛋白的结合常数有所变化,而结合位点数仍然为1。     

荧光光谱法分析米诺环素与人血清蛋白的相互作用

［摘要］目的研究不同温度下米诺环素与人血清蛋白（HSA）间的相互作用及其作用机制。方法通过荧光光谱法研究米诺环素对HSA的荧光猝灭光谱和同步荧光光谱,确定荧光猝灭的方式,并根据热力学方程讨论两者间主要的作用力类型。结果米诺环素对HSA荧光呈规律性猝灭,T＝299 K时,Ksv为3.29×104 L&#8226;mol-1,T＝310 K,时Ksv为3.53×104 L&#8226;mol-1。米诺环素使清蛋白的同步光谱中最大发射峰红移。 结论米诺环素对HAS的猝灭机制属于动态猝灭,两者之间的作用力以疏水作用力为主。     

发酵液中重组人血清白蛋白的纯化

采用摇瓶培养重组毕赤氏酵母（Pichia pastoris）表达并分泌重组人血清白蛋白（recombinant human se-rum alburmin，rHSA）至胞外，发酵液经（NH4）2SO4沉淀、离子交换层析、疏水层析和凝胶过滤分离纯化rHSA，纯化样品经SDS-PAGE检测为单一条带。     

荧光光谱法研究Ca2+存在下左氧氟沙星与人血清白蛋白的结合作用

目的：研究左氧氟沙星对人血清白蛋白以及在ca^2＋存在下左氧氟沙星与人血清白蛋白的结合作用。方法：通过荧光光谱法分析了左氧氟沙星对人血清白蛋白以及ca^2＋存在条件下左氧氟沙星对人血清白蛋白荧光淬灭光谱、同步荧光光谱,根据热力学方程讨论两者间主要的作用力类型。结果：在生理条件（pH=7．4,37℃）下,根据Stem—Volmer方程和荧光淬灭双倒数图,确定了左氧氟沙星对人血清白蛋白淬灭类型为静态淬灭,左氧氟沙星对人血清白蛋白的结合常数K=1．46×10^5L·mol^-1,结合位点n=1．1,根据热力学方法确定作用力类型为疏水作用力;在ca^2＋存在条件下,淬灭类型和作用力类型不变,结合常数K=2．38×10^4L·mol^-1,结合位点n：1．02。结论：在ca^2＋存在条件下,左氧氟沙星对人血清白蛋白的荧光淬灭减弱,结合常数和结合位点均变小。为研究左氧氟沙星的生物学效应,以及左氧氟沙星和Ca^2＋对蛋白质构象的影响等提供了重要信息。     

棉酚与人血清白蛋白相互作用及其分子模拟研究

摘 要 目的： 探讨棉酚与人血清白蛋白(HSA)的相互作用。方法: 采用荧光光谱方法研究在生理条件下,棉酚与人血清白蛋白（HSA）的相互作用,并采用分子对接软件模拟棉酚与人血清白蛋相互作用。结果:棉酚与HSA的结合常数为2.390 6×10⁵ L·mol^-1 (293K) 和3.576 8×10³ L·mol^-1（303K）, 具有一个结合位点,结合反应的主要作用力为氢键和范德华力,结合位置更接近于人血清白蛋白的酪氨酸残基,分子模拟分析也证实此结果。结论: 棉酚对人血清白蛋白的荧光猝灭机制属于静态荧光猝灭。     

Binding Study of the Fluorescence Probe l-Anilino-8-naphthalenesulfonate to Human Plasma and Human and Bovine Serum Albumin Using Potentiometric Titration

The binding of l-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA), human serum albumin (HSA), and human plasma has been studied by potentiometric titration utilizing a laboratory constructed ion selective electrode (ISE) of ANS. Three classes of ANS binding sites were found on BSA, HSA, and plasma at 25 and 37°C. Computer analysis of the data resulted in estimates for the association constants, number of binding sites (HSA, BSA), and binding capacity of each class. The association constants for the first class of binding sites at 25°C were found to be 7.53 (±0.59) × 10⁵, 2.70 (±0.20) × 10⁵, and 2.64 (±0.26) × 10⁵ M ^–l for BSA, HSA, and plasma, respectively. Lower values for the association constants of all binding classes were estimated at the higher temperature (37°C). The binding capacity for ANS decreased in the order BSA, plasma, HSA.     

荧光光谱法研究灯盏花素与牛血清白蛋白相互作用

目的 采用荧光光谱法研究灯盏花素与牛血清白蛋白(BSA)在生理条件下(pH 7.4)的相互作用机制。 方法 固定BSA浓度,依次加入不同浓度的灯盏花素溶液,在激发波长为280 nm下,290~500 nm的波长范围内进行荧光猝灭光谱、同步荧光光谱扫描。 结果 灯盏花素对BSA的荧光猝灭类型是静态猝灭;在温度288 K和310 K时,二者的结合常数KA分别为8.295×10⁵和3.302×10⁵ L·mol^-1;二者的结合位点数n分别为1.239 3和1.177 0。由热力学参数焓变(ΔH=-31.080 kJ·mol^-1)小于零和熵变(ΔS=5.392 J·mol^-1·K^-1)大于零,确定灯盏花素与BSA之间的作用力类型为静电作用力;生成自由能变(ΔG)为负值,表明灯盏花素与BSA的作用过程是一个自发过程。此外,应用同步荧光光谱考察了灯盏花素对BSA构象的影响。 结论 经过荧光光谱分析,可以确定灯盏花素与白蛋白的作用机制,为其开发成治疗心血管疾病新药提供理论依据。     

Binding and Anticancer Properties of Plumbagin with Human Serum Albumin

Plumbagin has received extensive attention as a promising anticancer drug. Therefore, we investigated the binding and anticancer properties of plumbagin with human serum albumin. Fluorescence results demonstrated that plumbagin interacts with human serum albumin, although its binding affinity may be affected to various extents by different compounds. The human serum albumin–plumbagin complex structure revealed that plumbagin binds to the hydrophobic cavity in the IIA subdomain of human serum albumin through hydrogen bonding and hydrophobic interactions. The plumbagin–human serum albumin complex enhances cytotoxicity by 2‐ to 3‐fold particularly in cancer cells but has no effect on normal cells in vitro. Compared with the unbound drug, the human serum albumin–plumbagin complex promotes HeLa cell apoptosis and has a stronger capacity for cell cycle arrest at the G2/M phase of HeLa cells. In conclusion, this study contributes to the rational design and development of plumbagin‐based drugs and a drug–human serum albumin delivery system.     

荧光光谱法分析盐酸哌唑嗪与牛血清白蛋白的结合作用

目的研究不同温度下盐酸哌唑嗪与牛血清白蛋白(BSA)间的相互作用及其作用机制。方法荧光光谱法。结果荧光光谱法测定盐酸哌唑嗪与BSA在25℃和37℃反应的结合常数K均是2.0×104L.mol-1,结合位点数n分别为1.19,1.14,热力学参数为(37℃)ΔH=0,ΔG=-25.52kJ.mol-1,ΔS=0.082kJ.mol-1.K-1,盐酸哌唑嗪的加入影响了BSA的构象;依据Frster非辐射能量转移机制,得到盐酸哌唑嗪与BSA之间的结合距离和能量转移效率分别为r=4.69nm,E=0.078。结论盐酸哌唑嗪与BSA在实验浓度范围内形成了具有一定结构的复合物,且它们之间的作用力以静电作用力为主。     

加替沙星与牛血清白蛋白相互作用的研究

目的研究不同酸度条件下, 加替沙星与牛血清白蛋白之间的相互作用.方法采用荧光光谱和紫外光谱法进行研究.结果运用荧光猝灭双倒数图计算了在不同条件下二者的结合常数K, 根据Foster非辐射理论计算在正常生理条件下二者的结合距离r, 并通过热力学参数确定了二者的作用力类型.结论加替沙星与牛血清白蛋白之间有较强的相互作用, 以电荷作用力为主.     

Effect of Oxidative Stress on the Structure and Function of Human Serum Albumin

Purpose. Human serum albumin (HSA) was mildly oxidized by a metal–catalyzed oxidation system (MCO–HSA), chloramine–T (CT–HSA) or H₂O₂ (H₂O₂–HSA), and the effects of these treatments on the structural, drug–binding and esterase–like properties were studied. Methods. Protein conformation was examined by calorimetric, chromatographic, electrophoretic and spectroscopic techniques. Drug binding was studied by ultrafiltration method, and esterase–like activity was determined using p–nitrophenyl acetate as a substrate. Results. Far–UV and near–UV CD spectra indicated that significant structural changes had occured as the result of treatment with MCO–HSA and CT–HSA but not with H₂O₂–HSA. However, SDS–PAGE analysis does not provide precise information on gross conformational changes such as fragmentation, cross–linking and SDS–resistant polymerisation. The results of differential scanning calorimetry, the fluorescence of the hydrophobic probe 1,1–bis–4–anilino–naphthalene–5,5–sulfonic acid and the elution time from a hydrophobic HPLC column indicated that MCO–HSA and CT–HSA in particular, have a more open structure and a higher degree of exposure of hydrophobic areas than unoxidized HSA. In all cases, high–affinity binding of warfarin remained unchanged for all the oxidized HSAs. However, high–affinity binding of ketoprofen to CT–HSA and, especially, MCO–HSA was diminished. In addition, the esterase–like activity of these proteins were all decreased to the same low level. Conclusions. Mild oxidation of HSA has no detectable effect on the binding of drugs to site I in subdomain IIA. In contrast, both the ligand binding property of site II and the esterase–like activity of oxidized HSAs are decreased, most probably due to conformational changes in subdomain IIIA.