Species Differences of Serum Albumins: III. Analysis of Structural Characteristics and Ligand Binding Properties During N-B Transitions

Purpose. The aim of this study was to investigate the characteristics of the structural transitions and changes in ligand binding properties of different albumins during the pH-dependent structural transition, often referred to as the N-B transition. Methods. Structural transitions were evaluated by means of spectrometry, differential scanning calorimetry and chemical modification. In addition, ligand binding properties were investigated using typical site-specific bound drugs (warfarin, phenylbutazone, ibuprofen and diazepam). Results. Conformational changes, including N-B transition, clearly occurred in albumins from all species used in this study. The conformational stabilities of all the albumins were clearly lost in the weakly alkaline pH range. This was probably the result of the destruction of salt bridges between domain I and domain III in the albumin molecule. In addition, the profiles of the ANS-induced fluorescence were different and could be classified into two patterns, suggesting that hydrophobic pockets in the albumin molecules were different for the different species. The data suggest that the amino acid residues responsible for the transitions were some of the His residues located in domain I. Further, the ligand binding properties of the albumins were slightly different but statistically significant. Conclusions. The overall mechanisms of the N-B transition may be similar for all the albumins, but its impact is considerably different among the species in terms of both structural characteristics and ligand binding properties. Furthermore, the transitions appear to be multi-step transitions.     

Species Differences of Serum Albumins: II. Chemical and Thermal Stability

Purpose. The chemical and thermal stability of five species of mammalian serum albumins (human, bovine, dog, rabbit, and rat) were investigated, and conformational stabilities were compared to obtain structural information about the different albumins. Methods. The chemical stability was estimated by using guanidine hydrochloride (GdnCl), and monitored by fluorometry and circular dichroism (CD). Thermal stability was evaluated by differential scanning calorimetry (DSC). Results. In human, bovine, and rat albumin, two transitions were observed when GdnCl-induced denaturation was monitored fluorometrically, indicating at least one stable intermediate, although, in dog and rabbit albumin, only one transition was observed. However, GdnCl denaturation, as monitored by the ellipticity, showed a two-state transition in all species used in this study. Since these proteins, showing two transitions, contained a conserved tryptophan residue within domain II, these structural changes might have occurred in domain II during intermediate formation. DSC measurements showed that human, bovine, and rat albumin exhibited single sharp endotherms and these were clearly consistent with a two-state transition, while the deconvolution analysis of broad thermograms observed for dog and rabbit albumin showed that the absorption peaks could be approximated by a two-component composition, and were consistent with independent transitions of two different cooperative blocks. Conclusions. These experimental results demonstrate that species differences exist with respect to the conformational stability and the mechanism of the unfolding pathway for mammalian albumin.     

基于人血清白蛋白的药物递送系统研究进展

人血清白蛋白是血液中的一种天然转运蛋白,其生理功能主要是与各种内源性物质和外源性分子结合,然后转运到体内各种组织。人血清白蛋白的内部结合位点和表面活性基团使其能够同时携带多种分子进行多功能治疗。此外,在温和的条件下,人血清白蛋白还可以通过不同的方法被制备成载药纳米粒子。基于人血清白蛋白的药物递送系统因其具有生物降解性、非免疫原性、生物相容性和较长的半衰期等特点,近年来受到越来越多的关注。综述了基于人血清白蛋白的药物递送系统的最新研究进展,并提出了人血清白蛋白制剂临床转化面临的挑战和未来发展方向。     

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.     

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.     

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.     

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.     

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.     

Serum Protein Binding of Nonsteroidal Antiinflammatory Drugs: A Comparative Study

The unbound fraction in serum f _u , is a critical parameter in describing and understanding the pharmacokinetics of NSAIDs. We compared f _u for 6 different NSAIDs using ultrafiltration of pooled serum at pH 7.4 and 24C. Measurements covered a wide concentration range in order to define binding affinity and number of binding sites. HPLC was used to measure drug concentrations in serum and ultrafiltrate. Direct injection of ultrafiltrate and serum (diluted 250X) permitted quantitation down to approximately 70 nM for most of the NSAIDs, i.e., approximately 15–20 ng/ml. Assuming binding only to albumin, the data were fitted to a model of two classes of binding sites with dissociation constants K1 and K2. The lowest K1 (highest affinity) was found with flurbiprofen, 0.0658 M, the highest with ketoprofen, 5.23 M, an 80-fold difference. At low drug concentrations, f _u becomes virtually constant and approaches a lower limit, . The following values were calculated: diclofenac 0.21% fenoprofen 0.25%, flurbiprofen 0.022%, ketoprofen 0.52%, naproxen 0.039%, and tolmetin 0.37%. Thus the least bound NSAID, ketoprofen, had a value 24-fold that of the most highly bound, flurbiprofen. The NSAIDs also differed widely with regard to the extent of variation in f _u within the range of therapeutic concentrations, and hence with regard to their potential as displacers of other drugs.     

The Determination of Naproxen by Spectrofluorometry and its Binding to Serum Proteins

Abstract A fluorometric method for the determination of naproxen in serum, albumin solutions and protein free buffer solutions is described. The detection limit is about 10 ng/ml. Furosemide, thiopental and salicylic acid did not show any disturbing fluorescence while phenprocoumon did. The in vitro binding of naproxen in albumin solutions and serum was studied by equilibrium dialysis. A small but significant increase was found in the percentual binding in albumin solutions as compared to serum. The percentual binding was not affected by changes in the pH from 5-8. By fitting the binding data to a model assuming two classes of binding sites, association constants and binding capacities were determined. A very high affinity and a high capacity were found. The association constant for the first class of binding sites was higher for human serum albumin than for Serum (8.5 versus 5.3 · 10⁶ M^-1). The difference in the protein binding to the first class of binding sites in human serum albumin and serum can be explained by the existence of a competitive inhibitor in serum.     

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.     

左氧氟沙星和氧氟沙星与清蛋白的结合位点及荧光淬灭分析

摘要目的研究左氧氟沙星和氧氟沙星与人血清清蛋白的结合作用。方法通过荧光光谱法分析左氧氟沙星和氧氟沙星对人血清清蛋白荧光淬灭光谱,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,左氧氟沙星和氧氟沙星对人血清清蛋白淬灭类型为静态淬灭,左氧氟沙星对人血清清蛋白的结合常数K＝1.46×105 L•mol－1,结合位点n＝1.11,氧氟沙星对人血清清蛋白的结合常数K＝4.31×104 L•mol－1,结合位点n＝1.04,根据热力学方法确定作用力类型均为疏水作用力。结论与左氧氟沙星比较,氧氟沙星对人血清清蛋白的荧光淬灭减弱,结合常数和结合位点均变小,结合位置也有明显区别;这些数据给研究左氧氟沙星和氧氟沙星的药理作用和生物学效应,以及左氧氟沙星和氧氟沙星对蛋白质构象的影响等提供了重要信息。     

The in Situ Acetylation of an Immobilized Human Serum Albumin Chiral Stationary Phase for High-Performance Liquid Chromatography in the Examination of Drug–Protein Binding Phenomena

The in situ modification of an immobilized human serum albumin (HSA) high-performance liquid chromatographic chiral stationary phase by p-nitrophenyl acetate is reported. This procedure, which is thought to affect primarily a single reactive tyrosine residue within the protein structure, influenced the chromatographic retention and enantioselectivity factors of a wide range of solutes. For certain solutes, increases in both capacity factor and chiral resolution were observed. Ultrafiltration studies on representative test solutes using free HSA, treated in a similar manner to the immobilized protein, gave similar results as the chromatographic observations, indicating that the latter effects are not artifactual results of immobilization. The effect of the modification of HSA on the binding behavior of drugs reportedly sharing the site predominantly affected by the derivatization, namely, the indole–benzodiazepine binding site, varied greatly. This observation suggests that the affected binding area is not a single, tightly structurally defined site.     

Comparison of Binding Characterization of Two Antiviral Drugs to Human Serum Albumin

Ribavirin and lamivudine are representatives of antiviral drugs that are widely used to treat viral infections, especially chronic liver disease. To compare binding mechanism and behavior of antiviral drugs with human serum albumin (HSA), we performed fluorescence spectroscopy and X‐ray crystallography to investigate the interactions of ribavirin and lamivudine with HSA. Fluorescence spectroscopy showed ribavirin and lamivudine inhibit binding affinity each other. Our results further demonstrated that ribavirin and lamivdudine interaction with HSA could be affected by the presence of other compounds, including the non‐steroidal anti‐inflammatory drugs, indometacin. X‐ray structures revealed that ribavirin and lamivudine bind in IIA subdomain of HSA mainly by forming hydrogen bond and hydrophobic interactions forces. The carboxamido of ribavirin forms hydrogen bonds with Arg222; Hydroxyl group (6) of ribavirin forms hydrogen bond with Arg257. Hydroxyl group (15) of lamivudine forms hydrogen bond with Arg222; amino group (4) of lamivudine forms hydrogen bond with carbonyl of Arg257. Our results reveal the key biochemical and structural characteristics of the HSA interaction with ribavirin and lamivudine, providing guidance for future development of ribavirin‐ and lamivudine‐based compounds and a drug‐HSA delivery system.     

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.     

Serum trace elements in animal models and human depression. Part I. Zinc

In the present study we report the results of investigations into the serum zinc levels in a clinical study of 19 patients with unipolar depression; 16 normal controls and in three animal models of depression: chronic severe stress (CSS), chronic mild stress (CMS) and olfactory bulbectomy (OB) in rats. CSS model and unipolar depressed patients exhibit lower serum zinc levels than the appropriate controls. There was no effect on the value in CMS or OB models. The present findings confirm previous clinical data indicating decreased serum zinc levels in human depression. Moreover, the data indicate that differences exists in the serum zinc levels in different animal models of depression and suggest a similarity between the CSS model and human depression with regard to this marker. Copyright © 1999 John Wiley & Sons, Ltd.     

Binding of Digitoxin to Human Serum Proteins: Influence of pH on the Binding of Digitoxin to Human Albumin

Abstract: The binding of digitoxin to albumin was studied under various conditions with regard to electrolytes and pH. Considerable variations of Na, K, Ca and Mg within the range of clinical relevance did not influence the binding of digitoxin to albumin. Under the influence of excessive concentrations of digitoxin this binding followed the simple law of mass action with an average number of binding sites of about 0.5 independent of pH. The intrinsic association constants were about twice the apparent association constants, calculated in another way and with the assumption of one binding site per molecule of albumin. The binding of digitoxin to albumin was shown to depend on the pH of the medium with a maximum association constant at pH = 4.8 (intrinsic association constant, K = 5 x 10⁵ l/mol). The binding of digitoxin to human serum proteins was shown to be equal to that of the binding to albumin. Under conditions relevant for clinical interpretations the binding of digitoxin to other proteins was insignificant.     

The Effect of pH on Gallopamil Protein Binding

Pharmaceutical Research -     

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。