X‐ray Crystallographic and Fluorometric Analysis of the Interactions of Rhein to Human Serum Albumin

To investigate the interactions between natural drugs and human serum albumin (HSA), we performed fluorescence spectroscopy and X‐ray crystallography to gain insight into binding mechanism and behaviour of rhein to HSA. Our fluorescence results demonstrated that rhein strongly binds with HSA, and other compounds may affect binding affinity of rhein to different extent. Structural analysis revealed that rhein binds to the IIA subdomain of HSA. The carboxylate group of rhein forms hydrogen bonds with Arg218 and Lys199, as well as a salt bond with Arg222. Hydroxyl group (4) of rhein forms a hydrogen bond with His242, and hydroxyl group (5) of rhein forms a hydrogen bond with Arg257. Oxygen atom (7) of rhein forms a hydrogen bond with Arg222, and oxygen atom (6) of rhein forms a hydrogen bond with H₂O. Furthermore, hydroxyl group (4) of rhein also forms a hydrogen bond with H₂O. Our results reveal the biochemical and structural characteristics of the interaction between rhein and HSA, providing guidance for future development of rhein‐based compounds and a drug–HSA delivery system.     

Structural Basis of Non‐Steroidal Anti‐Inflammatory Drug Diclofenac Binding to Human Serum Albumin

Human serum albumin (HSA) is the most abundant protein in plasma, which plays a central role in drug pharmacokinetics because most compounds bound to HSA in blood circulation. To understand binding characterization of non‐steroidal anti‐inflammatory drugs to HSA, we resolved the structure of diclofenac and HSA complex by X‐ray crystallography. HSA‐palmitic acid–diclofenac structure reveals two distinct binding sites for three diclofenac in HSA. One diclofenac is located at the IB subdomain, and its carboxylate group projects toward polar environment, forming hydrogen bond with one water molecule. The other two diclofenac molecules cobind in big hydrophobic cavity of the IIA subdomain without interactive association. Among them, one binds in main chamber of big hydrophobic cavity, and its carboxylate group forms hydrogen bonds with Lys199 and Arg218, as well as one water molecule, whereas another diclofenac binds in side chamber, its carboxylate group projects out cavity, forming hydrogen bond with Ser480.     

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.     

Study of Competitive Displacement of Curcumin on α-zearalenol Binding to Human Serum Albumin Complex Using Fluorescence Spectroscopy

α-zearalenol (α-ZOL) is a mycotoxin with a strong estrogen effect that affects the synthesis and secretion of sex hormones and is transported to target organs through human serum albumin (HSA). Additionally, it has been reported that curcumin can also bind to HSA with high affinity at the same binding site as α-ZOL. Additionally, several studies reported that reducing the bound fraction of α-ZOL contributes to speeding up the elimination rate of α-ZOL to reduce its hazard to organs. Therefore, to explore the influence of a nutrition intervention with curcumin on α-ZOL effects, the competitive displacement of α-ZOL from HSA by curcumin was investigated using spectroscopic techniques, ultrafiltration techniques and HPLC methods. Results show that curcumin and α-ZOL share the same binding site (subdomain IIA) on HSA, and curcumin binds to HSA with a binding constant of 1.12 × 10⁵ M⁻¹, which is higher than that of α-ZOL (3.98 × 10⁴ M⁻¹). Ultrafiltration studies demonstrated that curcumin could displace α-ZOL from HSA to reduce α-ZOL’s binding fraction. Synchronous fluorescence spectroscopy revealed that curcumin could reduce the hydrophobicity of the microenvironment of an HSA–α-ZOL complex. This study is of great significance for applying curcumin and other highly active foodborne components to interfere with the toxicokinetics of α-ZOL and reduce its risk of its exposure.     

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.     

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.     

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。     

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.     

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。     

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.     

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。     

人血白蛋白注射液临床应用现状及合理使用策略研究

目的：评价首都医科大学附属北京同仁医院人血白蛋白的临床应用现状,探讨该药的合理使用策略,促进药物的合理应用。方法：回顾性调查我院临床科室2016年1-6月人血白蛋白的应用情况。结果：我院2016年1-6月有31个临床科室839人使用人血白蛋白,其中普外科用药人数居首,占总人数的20.26%,其次是心内科、胸外科、干保科、儿科病房,主要用于低蛋白血症的防治（59.50%）、心肺分流术、烧伤、血液透析的辅助治疗（9.00%）、肝硬化及肾病引起的水肿和腹水（4.50%）等;用药前血清白蛋白浓度在30 g·L^-1以下的病例占69.00%,35 g·L^-1以上的占16.00%,且有5.00%诊断为低蛋白血症的病例在用药前并未检查血清白蛋白水平。人血白蛋白用药疗程1~30天,平均疗程为（3.73±1.97）天;个人用药总量以10~30 g居多,其患者例数占总病例数的30.00%,其次是40~60g,占24.00%。结论：我院人血白蛋白使用的问题主要集中在用药指证不明确、用药选择不合理、用药疗程偏长、与其他静脉药物联合使用等方面,提示我院对人血白蛋白的应用还存在一些误区,需加强对人血白蛋白的合理规范使用。     

荧光光谱法研究顺铂对表柔比星与人血白蛋白结合作用的影响

目的研究顺铂对表柔比星与人血清白蛋白(HSA)结合作用的影响。方法通过荧光光谱法研究顺铂和表柔比星对HSA的荧光猝灭光谱,同步荧光光谱。由Lineweaver-Burk双倒数作图法确定反应的解离常数,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,顺铂和表柔比星与HSA都有荧光猝灭作用。顺铂、表柔比星对HSA的猝灭过程为静态猝灭。表柔比星与HSA的结合点数为1,主要作用力为疏水作用力。顺铂不影响表柔比星对HSA的内源荧光猝灭作用,但能增加表柔比星与HSA的结合常数(KA)。结论顺铂不影响表柔比星的血药浓度,但能增加表柔比星与HSA的结合力。     

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。     

Interaction of Benzothiadiazides with Human Serum Albumin Studied by Dialysis and Spectroscopic Methods

The interaction of a series of benzothiadiazides with human serum albumin (HSA) was investigated by equilibrium dialysis (ED) and spectroscopic methods including circular dichroism (CD). The primary binding site of benzothiadiazides was designated site II, the diazepam site on the HSA molecule, as indicated by displacement experiments using different site-selective probes. Tyrosine and lysine amino acid residues were probably involved in the binding site of these compounds to HSA. Both electrostatic and hydrophobic interactions were found to play a role in the binding of these compounds to HSA. Among the compounds tested, chlorothiazide had the highest affinity (K₁ = 5.5 × 10⁴M^–1, K₂ = 5.8 × 10³ M^–1).The primary binding affinity of the compounds for HSA was of the order: chlorothiazide > cyclopenthiazide > polythiazide > ethiazide > trichlormethiazide = methyclothiazde > hydrochlorothiazide. Binding was insensitive to the N-B transition of HSA. The binding site is proposed to consist of a cationic site on the surface of the HSA molecule with a hydrophobic crevice to accommodate the aromatic ring of the compounds. Positions 3 and 7 of the benzothiadiazide molecule is thought to affect the binding affinity to HSA.     

Stability of Human Serum Albumin During Bioprocessing: Denaturation and Aggregation During Processing of Albumin Paste

Purpose. To assess the impact of various bioprocessing steps on thestability of freshly precipitated human serum albumin (HSA) obtainedfrom pooled human plasma.Methods. After initial precipitation of HSA from plasma, the resultantpaste is either (a) lyophilized or (b) washed with acetone and thenair-dried in order to obtain a dry powder. The structure of HSA wasexamined using Fourier transform infrared (IR) spectroscopy. Theextent of aggregation of redissolved HSA was measured using bothdynamic light scattering and SDS-polyacrylamide gel electrophoresis(SDS-PAGE).Results. Both lyophilization and air-drying perturb the secondarystructural composition of HSA, as detected by infrared (IR) spectroscopy.Upon dissolution of dried paste, most of the protein refolds to anative-like conformation. However, a small fraction of the protein moleculesform soluble aggregates that can be detected by both dynamic lightscattering and SDS-PAGE. The level of aggregation is so low that itcould not be detected in the bulk by either circular dichroism orIR spectroscopy. The lyophilized protein, which appears to be moreunfolded in the solid state than the acetone washed/air-dried material,exhibits a higher level of aggregation upon dissolution.Conclusions. There is a direct correlation between the extent ofunfolding in the solid state and the amount of soluble aggregate presentafter dissolution. Moreover, the presence of the aggregates persiststhroughout the remainder of the purification process, which includesdissolution, chromatography, sterile filtration and viral inactivationsteps. Analytical methods used to monitor the stability ofbiopharmaceuticals in the final product can be used to assess damage inflictedduring processing of protein pharmaceuticals.     

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.     

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.     

人血白蛋白278例临床应用调查与分析

目的：分析我院人血白蛋白的临床应用情况,促进其合理应用。方法：利用美康合理用药临床药学工作站,收集2009年4—6月我院9个内科科室和4个外科科室共278例人血白蛋白使用病例,采用Excel2003对其年龄、科室、疾病种类等用药情况进行统计分析。结果：278例患者中,外科用量大于内科,以肝胆外科分布最多,占总例数的22．30％,其次为消化内科（14．75％）、心血管外科（11．87％）、普外科（10．79％）;个人用药量多为10-20g;主要用于肿瘤、癌症（82例）、心脏疾病（36例）及骨关节疾病（23例）等重症患者;使用前患者血清白蛋白浓度主要集中在26—35g·L^-1范围内。结论：我院人血白蛋白的临床应用基本合理,但还存在一些使用误区,应本着有效、安全、经济的合理用药原则,严格掌握白蛋白的适应证,促进人血白蛋白的合理应用。