分子模拟和光谱法研究华法林与人血清白蛋白的结合机制

华法林(WF)是临床常用口服抗凝药物,有关它与人血清白蛋白(HSA)结合位点处的热点残基、作用力类型以及对HSA构象影响报道较少.通过在模拟人体生理条件下,利用分子模拟、内源荧光光谱、三维荧光光谱和同步荧光光谱,研究了WF与HSA之间作用机理.WF能使HSA构象发生改变,导致Tyr残基微环境极性降低、Trp残基周围亲水...     

Electrochemical Studies of Camptothecin and Its Interaction with Human Serum Albumin

Camptothecin, an anticancer component from Camptotheca acuminate, may interact with human serum albumin (HSA) at the subdomain IIA (site I), and then convert to its inactive form(carboxylate form). In this paper, the detailed electrochemical behaviors of camptothecin at a pyrolytic graphite electrode is presented. The interaction between camptothecin and HSA is also studied by electrochemical technique. By comparing with bovine serum albumin (BSA), which is highly homologous to HSA, we prove that camptothecin can specifically bind to HSA. Meanwhile, the inhibitory influence of sodium salicylate to this binding is also discussed.     

Spectroscopic and Molecular Docking Investigation on the Interaction of Cumin Components with Plasma Protein: Assessment of the Comparative Interactions of Aldehyde and Alcohol with Human Serum Albumin

The interaction of the important plasma protein, human serum albumin (HSA), with two monoterpenes found in cumin oil, i.e., cuminaldehyde (4-isopropylbenzaldehyde) and cuminol (4-isopropylbenzyl alcohol), was studied in this paper. Both experimental and computational methods were utilized to understand the mechanism of binding. The UV absorption profile of HSA changes in the presence of both cuminaldehyde and cuminol, due to the interaction between HSA with both monoterpenes. The intrinsic fluorescence intensity of HSA was also quenched on the sequential addition of both ligands, due to change in the microenvironment of the fluorophore present in the former. Quenching of HSA by cuminaldehyde was much higher in comparison to that in the presence of cuminol. Fluorescence quenching data were analyzed using modified Stern-Volmer and Lineweaver-Burk methods, which suggested that the binding mechanism was of a static type for both ligands. In both cases, the binding was favored by the domination of hydrophobic as well as hydrogen bonding/Van der Waals forces. Both ligands partially unfolded the secondary structure of HSA, although the effect of cuminaldehyde was more pronounced, as compared to cuminol. The preferred binding site of cuminaldehyde and cuminol inside HSA was also the same; namely, drug binding site 1, located in subdomain IIA. The study showed that cuminaldehyde binds strongly with albumin as compared to its alcohol counterpart, which is due to the more hydrophobic nature of the former.     

重组人血白蛋白与小分子药物结合功能研究

目的研究重组人血白蛋白与小分子药物的结合功能。方法在366nm下检测地西泮与人血白蛋白结合前后吸收值的变化,计算二者的结合常数;华法令与人血白蛋白结合后,用SephadexG-25HPLC柱进行分离,对游离华法令和二者结合产物分别积分,得到华法令与人血白蛋白的结合率。结果重组人血白蛋白与地西泮的结合常数为14·68mol Diaze-pam/mol rHSA,天然人血白蛋白与地西泮结合常数为12·82mol Diazepam/mol HSA。重组人血白蛋白中华法令的结合率为8·69%,而天然人血白蛋白中华法令的结合率为6·73%。结论重组人血白蛋白与天然人血白蛋白小分子药物结合功能基本一致。     

Noncovalent Association Thermodynamics of Turn-On Fluorescent Probes with Human Serum Albumin: Dual-Concentration Ratio Method

Efficient quantification of the affinity of a drug and the targeted protein is critical for strategic drug design. Among the various molecules, turn-on fluorescent probes are the most promising signal transducers to reveal the binding strength and site-specificity of designed drugs. However, the conventional method of measuring the binding ability of turn-on fluorescent probes by using the fractional occupancy under the law of mass action is time-consuming and a massive sample is required. Here, we report a new method, called dual-concentration ratio method, for quantifying the binding affinity of fluorescent probes and human serum albumin (HSA). Temperature-dependent fluorescence intensity ratios of a one-to-one complex (L ⋅ HSA) for a turn-on fluorescent probe (L), e. g., ThT (thioflavin T) or DG (dansylglycine), with HSA at two different values of [L]₀/[HSA]₀ under the constraint [HSA]₀>[L]₀ were collected. The van't Hoff analysis on these association constants further resulted in the thermodynamic properties. Since only two samples at different [L]₀/[HSA]₀ are required without the need of [L]₀/[HSA]₀ at a wide range, the dual-concentration ratio method is an easy way to greatly reduce the amounts of fluorescent probes and proteins, as well as the acquisition time.     

Serotonin Promotes Serum Albumin Interaction with the Monomeric Amyloid β Peptide

Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aβ by a factor of 3 (BBRC, 510(2), 248–253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aβ monomer to HSA by a factor of 7/17 for Aβ₄₀/Aβ₄₂, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA’s affinity to monomeric Aβ, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aβ release from HSA in the central nervous system due to impairment of the SRO-mediated Aβ trapping by HSA.     

Characterisation of cisplatin binding sites in human serum proteins using hyphenated multidimensional liquid chromatography and ESI tandem mass spectrometry

Cisplatin binding sites in human serum proteins have been characterised by using combined multidimensional liquid chromatography and ESI tandem mass spectrometry (MudPIT). Following incubation periods of 3 h for cisplatin-blood serum mixtures and subsequent trypsin digestion, MS-MS spectra were recorded for individual peptides that had been separated by SCX and RP liquid chromatography. Matching of the MS-MS spectra to theoretical sequences that were generated for human proteins in the SWISS-PROT database led to the identification of specific binding sites in human serum albumin (HSA), serotransferrin (Trfe) and other abundant serum proteins (A2mg, A1at, Apoa1, Apoa2). The cisplatin coordination sites in HSA and Trfe were confirmed by independent MudPIT studies on cisplatin reaction mixtures with the individual proteins. A total of five specific binding sites were identified for HSA, including the cysteine residue C34, two methionine sites (M329, M548) and the tyrosine and aspartate O-donor sites Y150 (or Y148) and D375 (or E376). Methionine-256 was established as a cisplatin coordination site for Trfe in addition to the O-donor sites E265, Y314, E385 and T457. Inspection of the protein structures indicates that the preferred residues belong either to peripheral alpha helices or to flexible loops within the protein-binding pockets. O-donor residues dominate as cisplatin binding sites for other abundant serum proteins.     

Investigation of the Binding Behavior between the S-heterocyclic Aromatic Palladium(II) Complex and Human Serum Albumin: Spectroscopic Approach

The interaction of 1, 10-phenanthroline octhyldithiocarbamato palladium(II) nitrate ([Pd(Oct-dtc)(phen)]NO₃) with human serum albumin (HSA) has been investigated by various spectroscopic techniques under physiological conditions. Here, HSA was titrated with the Pd(II) complex, followed by UV–Vis absorption spectroscopy to estimate a binding constant (K_b) and other thermodynamic parameters. The results indicate that the Pd (II) complex has a high affinity for bind HSA. Thermodynamic analysis showed that the enthalpy (ΔH°) and entropy changes (ΔS°) are positive and Gibbs free energy change (ΔG°) is negative which indicated that hydrophobic interactions played the predominant role in the binding process. Fluorescence spectroscopy were used to show the mechanism and binding parameters of this interaction. Utilizing the Stern–Volmer equation, the Pd(II) complex quenched the intrinsic fluorescence of HSA via a static quenching procedure. The specific binding distances between the tryptophan (donor) proteins and Pd(II) complex (acceptor) were estimated by Forster resonance energy transfer. The CD results also showed the conformational changes on serum albumin upon binding with the Pd(II) complex.     

Protein binding study of isoflavone, perillyl alcohol and S-ibuprofen by high-performance frontal analysis

High-performance frontal analysis (HPFA) was used for a protein binding study of isoflavones (daidzein, genistin, and genistein), enantiomers of perillyl alcohol and S-ibuprofen to human serum albumin (HSA). The analyses were performed on a Develosil and Inertsil 100-Diol-5 column (10 cm×4.6mm). Sodium phosphate solution (pH 7.4, ionic strength 0.17) was used as the mobile phase at a flow rate of 1 ml/min. To ensure the drug to be eluted as a trapezoidal peak with a plateau, injection volumes were each fixed up the zonal profile with an evident plateau appears. The unbound drug concentration was determined from a plateau height of the plateau region after that experimental data were fitted by Scatchard equation. The binding constants (K) and total binding affinities (nK) of drugs to HSA were calculated, respectively.     

Interaction of human serum album and c(60) aggregates in solution

An important property of C(60) in aquatic ecotoxicology is that it can form stable aggregates with nanoscale dimensions, namely nC(60). Aggregation allows fullerenes to remain suspended for a long time, and the reactivity of individual C(60) is substantially altered in this aggregate form. Herein, we investigated the interaction of nC(60) and human serum album (HSA) using the methods of fluorescence, fluorescence dynamics, circular dichroism (CD), and site marker competitive experiments. We proposed a binding model consistent with the available experimental results for the interactions of nC(60) with HSA. During the interaction process, the structure and conformation of HSA were affected, leading to functional changes of drug binding sites of HSA.     

Protein-based nanotubes for biomedical applications

This review presents highlights of our latest results of studies directed at developing protein-based smart nanotubes for biomedical applications. These practical biocylinders were prepared using an alternate layer-by-layer (LbL) assembly of protein and oppositely charged poly(amino acid) into a nanoporous polycarbonate (PC) membrane (pore diameter, 400 nm), with subsequent dissolution of the template. The tube wall typically comprises six layers of poly-L-arginine (PLA) and human serum albumin (HSA) [(PLA/HSA)(3)]. The obtained (PLA/HSA)(3) nanotubes (NTs) can be dispersed in aqueous medium and are hydrated significantly. Several ligands for HSA, such as zinc(II) protoporphyrin IX (ZnPP), were bound to the HSA component in the cylindrical wall. Similar NTs comprising recombinant HSA mutant, which has a strong binding affinity for ZnPP, captured the ligand more tightly. The Fe(3)O(4)-coated NTs can be collected easily by exposure to a magnetic field. The hybrid NTs bearing a single avidin layer as an internal wall captured biotin-labeled nanoparticles into the central channel when their particle size is sufficiently small to enter the pores. The NTs with an antibody surface interior entrapped human hepatitis B virus with size selectivity. It is noteworthy that the infectious Dane particles were encapsulated completely into the hollows. Other HSA-based NTs having an α-glucosidase inner wall hydrolysed a glucopyranoside to yield α-D-glucose. A perspective of the practical use of the protein-based NTs is also described.     

抗生素痢菌净与人血清白蛋白的相互作用

用荧光、同步荧光、三维荧光和紫外-可见吸收光谱法,研究了在生理条件下痢菌净(MEQ)与人血清白蛋白(HSA)相互作用的光谱学行为。分析了MEQ对HSA的荧光猝灭机制,计算了不同温度下的猝灭常数KSV、结合常数Kb、结合位点数n、热力学参数(如:ΔH、ΔS、ΔG)和结合距离r,并探研了MEQ对HSA构象的影响。结果表明:MEQ对HSA荧光猝灭方式是动态猝灭;结合位点数1;结合距离4.2 nm;MEQ对HSA构象的影响不大。MEQ与HSA间结合作用较弱,作为肠、胃或体外消炎药时,不易被血清白蛋白储存和转运,药物在体内的残留量较少、残留时间较短、所产生的副作用也较小。     

Spectrofluorometric and Molecular Docking Studies on the Binding of Curcumenol and Curcumenone to Human Serum Albumin

Curcumenol and curcumenone are two major constituents of the plants of medicinally important genus of Curcuma, and often govern the pharmacological effect of these plant extracts. These two compounds, isolated from C. zedoaria rhizomes were studied for their binding to human serum albumin (HSA) using the fluorescence quench titration method. Molecular docking was also performed to get a more detailed insight into their interaction with HSA at the binding site. Additions of these sesquiterpenes to HSA produced significant fluorescence quenching and blue shifts in the emission spectra of HSA. Analysis of the fluorescence data pointed toward moderate binding affinity between the ligands and HSA, with curcumenone showing a relatively higher binding constant (2.46 × 10⁵ M⁻¹) in comparison to curcumenol (1.97 × 10⁴ M⁻¹). Cluster analyses revealed that site I is the preferred binding site for both molecules with a minimum binding energy of −6.77 kcal·mol⁻¹. However, binding of these two molecules to site II cannot be ruled out as the binding energies were found to be −5.72 and −5.74 kcal·mol⁻¹ for curcumenol and curcumenone, respectively. The interactions of both ligands with HSA involved hydrophobic interactions as well as hydrogen bonding.     

Colloidal Fluorophore Aggregates for the Selective Detection of Albumins in Solution and on Electrophoresis Gels

We report a fluorescent probe that is highly sensitive and selective for serum albumins. Signal transduction results from the disassembly of fluorescence-quenched aggregates upon binding to human serum albumin (HSA). The probe offers a rapid (≤2 s) fluorometric assay of HSA in phosphate-buffered saline, urine, or blood serum with a high signal-to-noise ratio. It is also useful as a wash-free prestaining reagent for detecting HSA on electrophoresis gels.     

Quenching of Protein Fluorescence by Fullerenol C60(OH)36 Nanoparticles

The effect of the interaction between fullerenol C₆₀(OH)₃₆ (FUL) and alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae and human serum albumin (HSA) was studied by absorption spectroscopy, fluorescence spectroscopy, and time-resolved fluorescence spectroscopy. As shown in the study, the fluorescence intensities of ADH and HSA at excitation wavelengths λ_ex = 280 nm (Trp, Tyr) and λ_ex = 295 nm (Trp) are decreased with the increase in the FUL concentration. The results of time-resolved measurements indicate that both quenching mechanisms, dynamic and static, are present. The binding constant K_b and the number of binding sites were obtained for HSA and ADH. Thus, the results indicated the formation of FUL complexes and proteins. However, the binding of FUL to HSA is much stronger than that of ADH. The transfer of energy from the protein to FUL was also proved.     

CdTe/CdS量子点对木犀草素与人血清白蛋白结合力的影响

用还原型谷胱甘肽(GSH)作硫源和稳定剂在水相中合成了生物相容性好、量子产率高的核壳型CdTe/CdS半导体量子点;同时模拟人体内生理环境,研究了CdTe/CdS量子点(QDs)对木犀草素与人血清白蛋白(HSA)相互作用的影响。结果表明,CdTe/CdS QDs可有规律地猝灭HSA荧光;在CdTe/CdS QDs存在条件下,木犀草素可使HSA荧光最大发射波长从341 nm红移到347 nm,红移程度明显大于无QDs存在时,同时木犀草素与HSA的表观结合常数和结合位点数均有增加,即CdTe/CdS QDs使木犀草素与HSA的结合力增强。     

Ibuprofen Favors Binding of Amyloid-β Peptide to Its Depot,Serum Albumin

The deposition of amyloid-β peptide (Aβ) in the brain is a critical event in the progression of Alzheimer’s disease (AD). This Aβ deposition could be prevented by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aβ. We show here that an exogenous HSA ligand, ibuprofen (IBU), exerts the analogous effect. Plasmon resonance spectroscopy data evidence that a therapeutic IBU level increases HSA affinity to monomeric Aβ₄₀/Aβ₄₂ by a factor of 3–5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aβ₄₀ fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aβ₄₀-binding sites of HSA. The revealed enhancement of the HSA–Aβ interaction by IBU and the strengthened inhibition of Aβ fibrillation by HSA in the presence of IBU could contribute to the neuroprotective effects of the latter, previously observed in mouse and human studies of AD.     

Acetylcholinesterase Reactivators (HI-6, Obidoxime,Trimedoxime, K027, K075, K127, K203, K282): Structural Evaluation of Human Serum Albumin Binding and Absorption Kinetics

Acetylcholinesterase (AChE) reactivators (oximes) are compounds predominantly targeting the active site of the enzyme. Toxic effects of organophosphates nerve agents (OPNAs) are primarily related to their covalent binding to AChE and butyrylcholinesterase (BChE), critical detoxification enzymes in the blood and in the central nervous system (CNS). After exposure to OPNAs, accumulation of acetylcholine (ACh) overstimulates receptors and blocks neuromuscular junction transmission resulting in CNS toxicity. Current efforts at treatments for OPNA exposure are focused on non-quaternary reactivators, monoisonitrosoacetone oximes (MINA), and diacylmonoxime reactivators (DAM). However, so far only quaternary oximes have been approved for use in cases of OPNA intoxication. Five acetylcholinesterase reactivator candidates (K027, K075, K127, K203, K282) are presented here, together with pharmacokinetic data (plasma concentration, human serum albumin binding potency). Pharmacokinetic curves based on intramuscular application of the tested compounds are given, with binding information and an evaluation of structural relationships. Human Serum Albumin (HSA) binding studies have not yet been performed on any acetylcholinesterase reactivators, and correlations between structure, concentration curves and binding are vital for further development. HSA bindings of the tested compounds were 1% (HI-6), 7% (obidoxime), 6% (trimedoxime), and 5%, 10%, 4%, 15%, and 12% for K027, K075, K127, K203, and K282, respectively.     

Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA) and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (K_a = approximately 10⁴ M⁻¹). CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (K_EB = approximately 10⁴ M⁻¹). Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches.     

Porous magnetic chelator support for albumin adsorption by immobilized metal affinity separation

Magnetic poly(2‐hydroxyethylmethacrylate) (mPHEMA) beads are modified by iminodiacetic acid (IDA) to implify the reactive groups and subsequent binding of Cu²⁺ ions to form metal chelate. mPHEMA beads, in the size range of 80–120 μm, were produced by a modified suspension polymerization technique. mPHEMA beads were characterized by swelling tests, electron spin resonance (ESR), FTIR, and scanning electron microscopy (SEM). Important results obtained in this study are as follows. The swelling ratio of mPHEMA beads was 34%. The presence of magnetite particles in the polymeric structure was confirmed by ESR. FTIR data confirmed that the magnetic polymer beads were modified with functional groups IDA. The mPHEMA beads have a spherical shape and porous structure. The effect of pH and concentration of human serum albumin (HSA), on the adsorption of HSA to the metal‐chelated magnetic beads, were examined in a batch reactor. Most importantly, the magnetic beads had little nonspecific adsorption for HSA (0.5 mg/g) before introducing IDA groups. Cu²⁺ chelation increased the HSA adsorption up to 28.4 mg/g. Adsorption behavior can be described at least approximately with the Langmuir equation. Regeneration of the metal‐chelated magnetic beads was easily performed with 1.0M NaSCN, pH 8.0, followed by washing with distilled water and reloading with Cu²⁺. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 2501–2510, 2004