HPLC chiral stationary phases produced with isolated human serum albumin fragments.

The enantioselectivity of HPLC chiral stationary phases produced with human serum albumin (HSA) fragments was investigated. An HSA fragment (HSA-FG75) was isolated by size-exclusion chromatography following peptic digestion of HSA. The isolated HSA-FG75 was mainly an N-terminal half peptide with an average molecular weight of about 35,000 daltons. The HSA and HSA-FG75 proteins were bound to aminopropylsilica gels activated by N,N'-disuccinimidyl carbonate. Though the HSA-FG75 column showed lower enantioselectivities for all of the racemates tested than the intact HSA column, the enantioseparations of the racemates tested were attained with a shorter analysis time on the HSA-FG75 column. These results are ascribable to removal of the non-specific binding sites of HSA, changes in the globular structure of the HSA fragment and/or changes in the local environment around the binding sites. Further, the HSA-FG75 column was as stable as the intact HSA column for repetitive injection of samples.     

Interaction of acrylodan with human serum albumin. A fluorescence spectroscopic study

The binding of the fluorescent probe acrylodan (AC) to human serum albumin (HSA) was studied by fluorescence spectroscopy. The binding isotherms could be fitted to two types of sites. Competition experiments using iodoacetamide suggested that AC binds tightly on HSA by the cysteine-34. Attempts were made to find the location of the second site using high concentrations of warfarin, phenylbutazone, diazepam, indomethacin, palmitic acid or bilirubin in order to displace the bound AC to the HSA. Bilirubin was the only ligand able to displace the bound AC. This result suggests that AC, which is a very hydrophobic molecule also capable of labeling lysine residues, should also bind the human albumin in the primary site of bilirubin, but with less affinity than to the cysteine-34.     

Detergent binding as a sensor of hydrophobicity and polar interactions in the binding cavities of proteins

To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein-ligand binding, we studied the interaction of human serum albumin (HSA) and beta-lactoglobulin with various aliphatic (C10-C14) cationic and zwitterionic detergents. We find that cationic detergents, at levels that do not cause unfolding, interact with a single site on beta-lactoglobulin and with two primary and five to six secondary sites on HSA with an affinity that is approximately the same as that with which zwitterionic (dimethylamineoxide) detergents interact, suggesting the absence of significant electrostatic interactions in the high-affinity binding of these compounds. The binding affinity for all of the groups of compounds was dependent upon hydrocarbon chain length, suggesting the predominant role of hydrophobic forces, supported by polar interactions at the protein surface. A distinct correlation between the binding energy and the propensity for micelle formation within the group of cationic or noncharged (nonionic and zwitterionic) detergents indicated that the critical micellar concentration (CMC) for each of these detergent groups, rather than the absolute length of the hydrocarbon chain, can be used to compare their hydrophobicities during their interaction with protein. Intrinsic fluorescence data suggest that the two primary binding sites on serum albumin for the zwitterionic and cationic compounds are located in the C-terminal part of the albumin molecule, possibly in the Sudlow II binding region. Comparisons with previous binding data on anionic amphiphiles emphasize the important contribution of ion bond formation and other polar interactions in the binding of fatty acids and dodecyl sulfate (SDS) by HSA but not by beta-lactoglobulin. Electrostatic interactions by cationic detergents played a significant role in destabilizing the protein structure at high binding levels, with beta-lactoglobulin being more susceptible to unfolding than HSA. Zwitterionic detergents, in contrast to the cationic detergents, had no tendency to unfold the proteins at high concentrations.     

Competitive Binding of Drugs to the Multiple Binding Sites on Human Serum Albumin: A calorimetric study

A multiple-site competitive model has been developed to evaluate quantitatively the equilibrium competition of drugs that bind to multiple classes of binding sites on human serum albumin (HSA). The equations, which are based on the multiple-class binding site model, assume that competition exists at individual sites, that the binding parameters for drug or drug competitor pertain to individual sites, and also that the binding parameters for drug or competitor at any given site are independent of drug or competitor bound at other sites. For the drug-competitor pairs, ethacrynic acid (EA) -caproic acid (C6), -lauric acid (C12), and -palmitic acid (C16), the reaction heat of EA binding to HSA was measured in the absence and the presence of fatty acids at the molar ratio of 3:1 with HSA at pH 7.4 and 37°C by isothermal titration microcalorimetry. The calorimetric titration data induced by the presence of fatty acids were directly compaired to the computer simulation curves by the corresponding multiple-site competititve models, which were precedently calculated from binding parameters of EA and fatty acids. In the case of EA-C12 or -C16 competitive binding, EA binding at the first and the second classes of binding sites on HSA were instantaneously inhibited by C12 or C16, resulting that the binding constant of the first class of binding sites of EA were decreased and that the second class of binding sites on HSA entirely disappeared. In the competition between EA and C6, the first class of binding sites of EA was diminished by C6, resulting in the decrease of the binding constants and the number of binding sites in the first class of EA, whereas, the second class of binding sites was unaffected. The multiple-site competitive model assuming site-site competition could be directly comparable to the calorimetric data and be suitable to account for the competitive processes for drugs bound to the multiple-class of binding sites on HSA.This revised version was published online in November 2005 with corrections to the Cover Date.     

Investigation of the behavior of HSA upon binding to amlodipine and propranolol: Spectroscopic and molecular modeling approaches

The interaction between human serum albumin (HSA) and two drugs - amlodipine and propranolol - was investigated using fluorescence, UV absorption and circular dichroism (CD) spectroscopy. In addition, the binding site was established by applying molecular modeling technique. Fluorescence data suggest that amlodipine will quench the intrinsic fluorescence of HSA; whereas propranolol enhances the fluorescence of HSA. The binding constants for the interaction of amlodipine and propranolol with HSA were found to be 3.63×10(5)M(-1) and 2.29×10(4)M(-1), respectively. The percentage of secondary structure feature of each one of the HSA-bound drugs, i.e. the α-helix content, was estimated empirically by circular dichroism. The results indicated that amlodipine causes an increase, and that propranolol leads to a decrease in α-helix content of HSA. The spectroscopic analysis indicates that the binding mechanisms of the two drugs are different from each other. The data obtained by the molecular modeling study indicated that these drugs bind, with different affinity, to different sites located in subdomain IIA and IIIA.     

Identification of drug-binding sites on human serum albumin using affinity capillary electrophoresis and chemically modified proteins as buffer additives

A technique based on affinity capillary electrophoresis (ACE) and chemically modified proteins was used to screen the binding sites of various drugs on human serum albumin (HSA). This involved using HSA as a buffer additive, following the site-selective modification of this protein at two residues (tryptophan 214 or tyrosine 411) located in its major binding regions. The migration times of four compounds (warfarin, ibuprofen, suprofen and flurbiprofen) were measured in the presence of normal or modified HSA. These times were then compared and the mobility shifts observed with the modified proteins were used to identify the binding regions of each injected solute on HSA. Items considered in optimizing this assay included the concentration of protein placed into the running buffer, the reagents used to modify HSA, and the use of dextran as a secondary additive to adjust protein mobility. The results of this method showed good agreement with those of previous reports. The advantages and disadvantages of this approach are examined, as well as its possible extension to other solutes.     

Study of donepezil binding to serum albumin by capillary electrophoresis and circular dichroism

The interaction between human serum albumin (HSA) and the acetylcholinesterase inhibitor donepezil, has been studied by means of capillary electrophoresis frontal analysis (CE/FA) and circular dichroism. CE/FA enabled rapid and direct estimation of the quantity of free donepezil present at equilibrium with a physiological level of serum albumin (600 mol L^–1). Application of Scatchard analysis enabled estimation of the binding parameters of HSA towards donepezil, such as association constant and number of binding sites on one protein molecule. Furthermore, due to enantioseparation ability shown by HSA on donepezil in CE mode, displacement experiments were carried out using ketoprofen and warfarin as coadditives to the HSA based running buffer. The addition of these compounds reduced the enantioresolution of donepezil by HSA only when used at high concentration. These data were confirmed and corroborated by circular dichroism (CD) experiments. Using CD, bilirubin was also applied as a ligand specific to site III of HSA. The observed behaviour suggested that donepezil could be considered a ligand with independent binding to sites I and II; although site III is not the highest affinity site, indirect interaction (i.e. cooperative binding) can be assumed.     

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.     

Covalent binding of human serum albumin and ovalbumin by chloramine-T and chemical modification of the proteins

The covalent binding of ³⁵S-chloramine-T to human resum albumin (HSA) and ovalbumin is described. At pH 6.5, up to 24 chloramine-T molecules were found to be covalently bound per molecule of HSA; with ovalbumin the binding was only 5–7 molecule per protein molecule. Binding was accompanied by extensive modification of methionine, cysteine, histidine, tyrosine and lysine. Three new peaks appeared in the amino acid profiles of the modified proteins; two were identified as 1-aminoadipic acid (oxidation of lysine) and 3-chlorotyrosine. The most sites for covalent binding are lysine residues.     

Evaluation of enantioselective binding of antihistamines to human serum albumin by ACE

The drug binding to plasma and tissue proteins is a fundamental factor in determining the overall pharmacological activity of a drug. HSA, together with alpha(1)-acid glycoprotein, are the most important plasma proteins, which act as drug carriers, with implications on the pharmacokinetic of drugs. Among plasma proteins, HSA possesses the highest enantioselectivity. In this paper, a new methodology for the study of enantiodifferentiation of chiral drugs with HSA is developed and applied to evaluate the possible enantioselective binding of four antihistamines: brompheniramine, chlorpheniramine, hydroxyzine and orphenadrine to HSA. This study includes the determination of affinity constants of drug enantiomers to HSA and the evaluation of the binding sites of antihistamines on the HSA molecule. The developed methodology includes the ultrafiltration of samples containing HSA and racemic antihistaminic drugs and the analysis of the free or bound drug fraction using the affinity EKC-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of antihistamines to HSA, the major plasmatic protein.     

Studies on the binding of paeonol and two of its isomers to human serum albumin by using microcalorimetry and circular dichroism

Interactions of paeonol and two of its isomers with human serum albumin (HSA) in buffer solutions (pH 7.0) have been investigated by calorimetry and circular dichroism. Heats of the interactions have been determined with isothermal titration microcalorimetry at 298.15 K. Data process has been based on the supposition that there are several independent classes of binding sites on each HSA molecule for molecules of each one of the drugs. The results obtained by using this supposition combined with Langmuir adsorption model show that there are two classes of such binding sites. The binding constant, changes of enthalpy, entropy, and Gibbs free energy are obtained, which show that the two classes of binding are mainly driven by enthalpy except that the first-class binding of Ace is predominantly driven by entropy. On the same class of binding site, the negative value of binding enthalpy decreases in the order of Pae, Hma, and Ace. The difference of thermodynamic data is caused by the different locations of substituent groups on aromatic benzene ring of guest molecules. Circular dichroism (CD) spectra show that the three isomers change the secondary structure of HSA. These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of HSA induced by the three isomers.     

Protective effects of small amounts of bis(2-ethylhexyl)sulfosuccinate on the helical structures of human and bovine serum albumins in their thermal denaturations

The protective effect of an anionic double-tailed surfactant, sodium bis(2-ethylhexyl)sulfosuccinate (AOT), on the structures of human serum albumin (HSA) and bovine serum albumin (BSA) in their thermal denaturations was examined by means of circular dichroism measurements. The structural changes of these albumins were reversible in the thermal denaturation below 50 degrees C, but became partially irreversible above this temperature. The effect was observed in the thermal denaturation above 50 degrees C. Although the helicity of HSA decreased from 66% to 44% at 65 degrees C in the absence of the surfactant, the decrement of it was restrained in the coexistence of AOT of extremely low concentrations. When the HSA concentration was 10 muM, the maximal protective effect appeared at 0.15 mM AOT. In the coexistence of the surfactant of this concentration, the helicity was maintained at 58% at 65 degrees C, increasing to the original value upon cooling to 25 degrees C. Beyond 0.15 mM AOT, the helicity sharply decreased until 3 mM AOT. A particular AOT concentration required to induce the maximal protective effect ([AOT]REQ) was examined at different HSA concentrations. [AOT]REQ shifted to higher values with an increase of the protein concentration. From the protein concentration dependences of [AOT]REQ, the maximal protection was estimated to require 8.0 and 5.0 AOT ions per a molecule of HSA and BSA, respectively. The AOT concentration, where the protective effect was observed, was too low to form its micelle-like aggregate. Then the protein structures might be stabilized by a cross-linking of surfactant monomers bound to specific sites. These specific sites might exist between a group of nonpolar residues and a positively charged residue located on several sets of amphiphilic helical rods in the proteins. Such a unique function of the double-tailed ionic surfactant is first presented by its characteristic nature as an amphiphilic material.     

Spectroscopic investigation of the interaction between human serum albumin and three organic acids

The interactions of human serum albumin (HSA) with sinapic acid (SA), gallic acid (GA) and shikimic acid (SI) were investigated by fluorescence and Fourier transformed infrared spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of GA at the molar ratio of drug to HSA ranging from 0.1 to 30, and their binding constant (K(A)) is 1.1x10(4) M(-1). While one HSA molecule combined with one or two molecule of SA at the molar ratio of drug to HSA ranging from 0.1 to 4.26 or 4.26 to 30, and their binding affinities (K(A)) are 1.92x10(3) M(-1) and 6.87x10(8) M(-1), respectively. There is no specific interaction between HSA and SI. Combining the curve-fitting results of infrared amide I and amide III bands, the alterations of protein secondary structures induced by drugs were estimated. The drug-protein combination brought gradual reductions of the protein alpha-helix structure with increasing the concentrations of SA and GA, but SI did not change the protein secondary structure. From the fluorescence and FT-IR results, the binding mode was discussed in relation to the structures of the organic acids.     

Revealing the ionization ability of binding site I of human serum albumin using 2-(2'-hydroxyphenyl)benzoxazole as a pH sensitive probe

The ability of site I of human serum albumin (HSA) to bind medium sized molecules is important for the distribution, metabolism, and efficacy of many drugs. Herein, we show that this binding site has the ionization ability that may alter the drug structure during the process of its delivery. We reveal this ability by employing 2-(2'-hydroxyphenyl)benzoxazole (HBO) as a pH sensitive probe. Binding of HBO in site I is studied here at physiological pH 7.2 using steady-state and lifetime spectroscopic measurements, molecular docking and molecular dynamics (MD) simulation methods. The complex photophysics of HBO and the unique fluorescence signature of its anionic form indicate that, upon binding with HSA, the molecule exists in equilibrium between the anionic and the syn-keto forms. The position of HBO inside the binding site was determined experimentally by measuring the fluorescence quenching of W214, the sole tryptophan residue in HSA. The ionization degree of HBO inside the binding site was estimated to be close to the ionization degree of HBO in an aqueous solution of pH 10. This was concluded by comparing the fluorescence behavior of bound HBO to that of HBO in different solvents and in aqueous solutions of different pH values. Molecular docking and MD simulations show that HBO binds in site I close to W214, confirming the experimental results, and pinpoint the dominant role of hydrophobic interactions in the binding site. The formation of the anionic form is proposed to be due to through-space interaction between the OH group of HBO and both R222 and I290 with a binding mode similar to that of warfarin in site I. Comparison of the results with those of HBO mixed with key amino acids in solution indicates the importance of through-space interaction in the formation of the anion, similar to enzymatic reactions.     

Determination of drug-protein interactions by combined microdialysis and high-performance liquid chromatography

Summary A simple and rapid method for determination of the parameters of the interaction between drugs and protein, including the association constant and the number of binding sites, has been developed by use of a microdialysis sampling technique combined with high-performance liquid chromatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazepine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as examples) were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. The microdialysis probe was the used to sample the mixed CBZ-HSA solution at a perfusion rate of 1 μL min⁻¹. The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance liquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; theRSD ofR was approximately 1.85%. The estimated association constant (K) and the number of the binding sites,n, on one molecule of HSA were 1.06×10⁴ M⁻¹ and 0.880, respectively, which is in good agreement with the literature values determined by high-performance frontal analysis. The potential use of microdialysis is also discussed.     

补骨脂素和异补骨脂素键合人血清白蛋白的比较

将互为同分异构体的两种植物药活性组分补骨脂素和异补骨脂素作为研究对象,利用荧光光谱、紫外光谱、圆二色谱及傅立叶变换红外光谱详细比较研究了这两种香豆素类化合物与人血清白蛋白(HSA)的键合作用.不同光谱的结果定性、定量地显示了HSA二级结构变化的程度.依据荧光滴定实验及Van′t Hoff公式求出了反应的热力学参数(ΔH和ΔS)的值.根据修正后的Stern-Volmer和Scatchard方程和荧光光谱数据分别求得不同温度(296,303,310及318 K)下药物与蛋白相互作用的结合常数及结合位点数;且根据F觟rster偶极-偶极能量转移理论,求得药物与HSA间的键合距离;利用竞争实验确定了药物在HSA上的键合位点为site II.从分子水平上揭示了这两种化合物与HSA相互作用的机制.     

Identification of ligands that target the HCV-E2 binding site on CD81

Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.     

Microdialysis-liquid chromatographic study on competitive binding of drugs to protein

A new method to determine the interaction between drug and protein has been developed by utilizing the technique of microdialysis sampling with the ketoprofen and the human serum albumin (HSA) as the model of drug and protein.Two kinds of binding sites of HSA to ketoprofen have been observed.The binding constants and number of binding sites obtained by the Scatchard equation are 0.799,3.18×106 mol-1 L and 2.15,2.01×105 mol-1 L,respectively The displacement binding of drugs to HSA has also been studied.The strong displacement of competitive binding of ibuprofen with ketoprofen to HSA was observed,which means that the primary binding site of HSA to ketoprofen and that to ibuprofen are the same.However,only a weaker displacement of warfarin for the association of ketoprofen with HSA was observed,which may suggest that the primary binding site of HSA to ketoprofen is different from that to warfarin.Such a displacement effect for competitive binding of drugs to HSA was explained by the displacement model i     

Design and characterization of libraries of molecular fragments for use in NMR screening against protein targets

We have designed four generations of a low molecular weight fragment library for use in NMR-based screening against protein targets. The library initially contained 723 fragments which were selected manually from the Available Chemicals Directory. A series of in silico filters and property calculations were developed to automate the selection process, allowing a larger database of 1.79 M available compounds to be searched for a further 357 compounds that were added to the library. A kinase binding pharmacophore was then derived to select 174 kinase-focused fragments. Finally, an additional 61 fragments were selected to increase the number of different pharmacophores represented within the library. All of the fragments added to the library passed quality checks to ensure they were suitable for the screening protocol, with appropriate solubility, purity, chemical stability, and unambiguous NMR spectrum. The successive generations of libraries have been characterized through analysis of structural properties (molecular weight, lipophilicity, polar surface area, number of rotatable bonds, and hydrogen-bonding potential) and by analyzing their pharmacophoric complexity. These calculations have been used to compare the fragment libraries with a drug-like reference set of compounds and a set of molecules that bind to protein active sites. In addition, an analysis of the overall results of screening the library against the ATP binding site of two protein targets (HSP90 and CDK2) reveals different patterns of fragment binding, demonstrating that the approach can find selective compounds that discriminate between related binding sites.