Protein binding study of catechin hydrate and genistein by high-performance frontal analysis

High-performance frontal analysis (HPFA) was used for the protein binding study of catechin hydrate and genistein to human serum albumin (HSA). The experiment was performed on a Develosil 100Diol-5 column, and sodium phosphate buffer (pH 7.4 and ionic strength of 0.17) was used as the mobile phase. The mixtures of the drug-HSA solution were directly injected into the HPFA column, the HSA was eluted first and the unbound drugs were eluted out as a trapezoidal peak with a plateau region. The unbound drug concentration was determined from a plateau height of the plateau region and the experimental data were fitted by Scatchard equation. The binding constants (K) and binding affinities (nK) of the drug to HAS were K=1.32×10⁴ (L mol⁻¹), nK=0.47×10⁴ (L mol⁻¹) for catechin hydrate, and K=5.17×10⁴ (L mol⁻¹), nK=2.14×10⁴ (L mol⁻¹) for genistein.     

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残基周围亲水性增强。WF能有效猝灭HSA内源荧光,猝灭机制属静态猝灭,通过Lineweaver-Burk方程求得两者间结合常数(K_A)为3.55×10⁵L/mol(310 K)、结合位点数(n)约为1。Van′t Hoff定律计算得到的热力学参数(ΔH=169.56 kJ/mol和ΔS=653.23 J/(mol·K)),说明WF与HSA之间的主要作用力为疏水作用力。根据F9rster′s能量转移定律确定,WF与HSA结合距离为2.87 nm。WF通过氢键和疏水作用力与HSA的亚域ⅡA中氨基酸残基进行作用,WF周围4.0?区域还存在ⅠB、ⅡB、ⅢA亚结构域的30个氨基酸残基。     

Functionalization of Human Serum Albumin by Tyrosine Click

Human serum albumin (HSA) is a promising drug delivery carrier. Although covalent modification of Cys34 is a well-established method, it is desirable to develop a novel covalent modification method that targets residues other than cysteine to introduce multiple functions into a single HSA molecule. We developed a tyrosine-selective modification of HSA. Three tyrosine selective modification methods, hemin-catalyzed, horseradish peroxidase (HRP)-catalyzed, and laccase-catalyzed reactions were performed, and the modification efficiencies and modification sites of the modified HSAs obtained by these methods were evaluated and compared. We found that the laccase-catalyzed method could efficiently modify the tyrosine residue of HSA under mild reaction conditions without inducing oxidative side reactions. An average of 2.2 molecules of functional groups could be introduced to a single molecule of HSA by the laccase method. Binding site analysis using mass spectrometry suggested Y84, Y138, and Y401 as the main modification sites. Furthermore, we evaluated binding to ibuprofen and found that, unlike the conventional lysine residue modification, the inhibition of drug binding was minimal. These results suggest that tyrosine-residue selective chemical modification is a promising method for covalent drug attachment to HSA.     

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.     

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。     

Interactive Association of Drugs Binding to Human Serum Albumin

Human serum albumin (HSA) is an abundant plasma protein, which attracts great interest in the pharmaceutical industry since it can bind a remarkable variety of drugs impacting their delivery and efficacy and ultimately altering the drug’s pharmacokinetic and pharmacodynamic properties. Additionally, HSA is widely used in clinical settings as a drug delivery system due to its potential for improving targeting while decreasing the side effects of drugs. It is thus of great importance from the viewpoint of pharmaceutical sciences to clarify the structure, function, and properties of HSA–drug complexes. This review will succinctly outline the properties of binding site of drugs in IIA subdomain within the structure of HSA. We will also give an overview on the binding characterization of interactive association of drugs to human serum albumin that may potentially lead to significant clinical applications.     

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.     

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。     

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.     

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.     

Development of polylactide microspheres for protein encapsulation and delivery

The development of injectable microparticles for protein delivery is a major challenge. We demonstrated the possibility of entrapping human serum albumin (HSA) and thrombin (Thr) in poly(ethylene glycol) (PEG)‐coated, monodisperse, biodegradable microspheres with a mean diameter of about 10 μm. In our earlier studies, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) analysis was used to characterize the surface of PEG‐coated, taxol‐loaded poly(lactic acid) (PLA) microspheres. An analysis by DRIFTS revealed that PEG was incorporated well on the PLA microsphere surface. An emulsion of protein (in water) and PLA dissolved in an acetone–dichloromethane (or acetone–chloroform) mixture were poured into an aqueous solution of PEG [or poly(vinyl alcohol) (PVA)] with stirring with a high‐speed homogenizer for the formation of microparticles. HSA recovery in microspheres ranged from 13 to 40%, depending on the solvent and emulsification systems used for the preparation. PLA dissolved in a dichloromethane/acetone system and albumin loaded via a PEG emulsification solution (PLA–PEG–HSA) showed maximum drug recovery (39.5%) and drug content (9.9%). Scanning electron microscopy revealed that PEG‐coated microspheres had less surface micropores than PVA‐based preparations. The drug‐release behavior of microspheres suspended in phosphate‐buffered saline exhibited a biphasic pattern. An initial burst release (30%) followed by a constant slow release for 20 days was observed for HSA and Thr from PLA–PEG microspheres. PEG‐coated PLA microspheres show great potential for protein‐based drug delivery. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1285–1295, 2002     

Aqueous Biphasic Hydroformylation Catalysed by Protein‐Rhodium Complexes

The water‐soluble complex derived from Rh(CO)₂(acac) and human serum albumin (HSA) proved to be efficient in the hydroformylation of several olefin substrates. The chemoselectivity and regioselectivity were generally higher than those obtained by using the classic catalytic systems like TPPTS‐Rh(I) (TPPTS=triphenylphosphine‐3,3′,3″‐trisulfonic acid trisodium salt). Styrene and 1‐octene, for instance, were converted in almost quantitative yields into the corresponding oxo‐aldehydes at 60 °C and 70 atm (CO/H₂=1) even at very low Rh(CO)₂(acac)/HSA catalyst concentrations. The possibility of easily recovering the Rh(I) compound makes the system environmentally friendly. The circular dichroism technique was useful for demonstrating the Rh(I) binding to the protein and to give information on the stability in solution of the catalytic system. Some other proteins have been used to replace HSA as complexing agent for Rh(I). The results were less impressive than those obtained using HSA and their complexes with Rh(I) were much less stable.     

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.     

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.     

Long-term release of chlorhexidine from dental adhesive resin system using human serum albumin nanoparticles

A functional dental adhesive resin system with antimicrobial properties was developed using human serum albumin (HSA) nanoparticles as the drug delivery carrier. HSA nanoparticles loaded with chlorhexidine (CHX) diacetate, a model antimicrobial drug, were prepared using a desolvation technique. The resulting CHX-loaded HSA nanoparticles were incorporated into a commercial methyl methacrylate (MMA)-based resin. The size of the nanoparticles ranged 50–300 nm, and the nanoparticles were dispersed homogeneously in the resin matrix. The CHX-loaded HSA nanoparticles showed an early release burst of ~20 % of the total CHX by day 5, followed by the sustained release of the remaining CHX over the next 20 days. In contrast, the resin matrix containing the HSA nanoparticles showed a sustained release of CHX without an early release burst in a 4-week immersion study. In the agar diffusion test, the resin matrix incorporating the CHX-loaded HAS nanoparticles showed a larger growth inhibition zone against Streptococcus mutans than the resin matrix alone, indicating that this delivery platform potently imparts antibacterial activity to the resins. These results also suggest that CHX, which inhibits the growth of oral bacteria, can be incorporated efficiently into the MMA-based resin matrix using HSA nanoparticles.     

Development of Cobalt-Binding Peptide Chelate from Human Serum Albumin: Cobalt-Binding Properties and Stability

Radioactive isotopes are used as drugs or contrast agents in the medical field after being conjugated with chelates such as DOTA, NOTA, DTPA, TETA, CyDTA, TRITA, and DPDP. The N-terminal sequence of human serum albumin (HSA) is known as a metal binding site, such as for Co²⁺, Cu²⁺, and Ni²⁺. For this study, we designed and synthesized wAlb12 peptide from the N-terminal region of HSA, which can bind to cobalt, to develop a peptide-based chelate. The wAlb12 with a random coil structure tightly binds to the Co(II) ion. Moreover, the binding property of wAlb12 toward Co(II) was confirmed using various spectroscopic experiments. To identify the binding site of wAlb12, the analogs were synthesized by alanine scanning mutagenesis. Among them, H3A and Ac-wAlb12 did not bind to Co(II). The analysis of the binding regions confirmed that the His3 and α-amino group of the N-terminal region are important for Co(II) binding. The wAlb12 bound to Co(II) with K_d of 75 μM determined by isothermal titration calorimetry when analyzed by a single-site binding model. For the use of wAlb12 as a chelate in humans, its cytotoxicity and stability were investigated. Trypsin stability showed that the wAlb12 − Co(II) complex was more stable than wAlb12 alone. Furthermore, the cell viability analysis showed wAlb12 and wAlb12 + Co(II) to be non-toxic to the Raw 264.7 and HEK 293T cell lines. Therefore, a hot radioactive isotope such as cobalt-57 will have the same effect as a stable isotope cobalt. Accordingly, we expect wAlb12 to be used as a peptide chelate that binds with radioactive isotopes.     

Poly(acrylamide‐allyl glycidyl ether) cryogel as a novel stationary phase in dye‐affinity chromatography

Poly(acrylamide‐allyl glycidyl ether) [poly(AAm‐AGE)] cryogel was prepared by bulk polymerization which proceeds in an aqueous solution of monomers frozen inside a glass column (cryo‐polymerization). After thawing, the monolithic cryogel contains a continuous polymeric matrix having interconnected pores of 10–100 μm size. Cibacron Blue F3GA was immobilized by covalent binding onto poly(AAm‐AGE) cryogel via epoxy groups. Poly(AAm‐AGE) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(AAm‐AGE) monolithic cryogel was 6.84 g H₂O/g cryogel. Poly(AAm‐AGE) cryogel containing 68.9 μmol Cibacron Blue F3GA/g was used in the adsorption/desorption of human serum albumin (HSA) from aqueous solutions and human plasma. The nonspecific adsorption of HSA was very low (0.2 mg/g). The maximum amount of HSA adsorption from aqueous solution in acetate buffer was 27 mg/g at pH 5.0. Higher HSA adsorption value was obtained from human plasma (up to 74.2 mg/g). Desorption of HSA with a purity of 92% from Cibacron Blue F3GA attached poly(AAm‐AGE) cryogel was achieved using 0.1M Tris/HCl buffer containing 0.5M NaCl. It was observed that HSA could be repeatedly adsorbed and desorbed with poly(AAm‐AGE) cryogel without significant loss in the adsorption capacity. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Human serum albumin (HSA) adsorption with chitosan microspheres

In this study, chitosan microspheres were prepared and characterized for adsorption of human serum albumin (HSA) as affinity sorbent. The chitosan microspheres were obtained with a “suspension crosslinking technique” in the size range of 30–700 μm by using a crosslinker, i.e., glutaraldehyde. The chitosan microspheres used in HSA adsorption studies were having the average size of 170 ± 81 μm. Adsorption medium pH and the initial HSA concentration in the adsorption medium were changed as 4.0–7.0 and 0.5–2.0 mg HSA/mL, respectively, to investigate the HSA adsorption capacity of chitosan microspheres. Maximum HSA adsorption (i.e., 11.35 mg HSA/g chitosan microspheres) was obtained at pH 5.0 and 1.5 mg HSA/mL of the initial HSA concentration in the adsorption medium was obtained as the saturation value for HSA adsorption. A very common dye ligand, i.e., Cibacron Blue F3GA was attached to the chitosan microspheres to increase the HSA adsorption capacity. Actually, the HSA adsorption capacity was increased up to 15.35 mg HSA/g chitosan microspheres in the case of Cibacron Blue F3GA attached to chitosan microspheres used. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 3035–3039, 2002     

Preparation of a novel metal‐chelate affinity beads for albumin isolation from human plasma

In this study, we developed a novel approach to obtain a high protein‐adsorption capacity utilizing 2‐methacryloylamidohistidine (MAH) as a biollgand. MAH was synthesized by reacting methacryloyl chloride and histidine. Spherical beads, with an average size of 150–200 μm, were obtained by the radical suspension polymerization of MAH, ethyleneglycol dimethacrylate (EGDMA), and 2‐hydroxyethyl methacrylate (HEMA) conducted in an aqueous dispersion medium. p(EGDMA–HEMA–MAH) beads had a specific surface area of 17.6 m²/g. The synthesized MAH monomer was characterized by NMR. p(EGDMA–HEMA–MAH) beads were characterized by a swelling test, FTIR, and elemental analysis. Then, Cu(II) ions were incorporated into the beads and Cu(II) loading was found to be 0.96 mmol/g. These beads, with a swelling ratio of 65% and containing 1.6 mmol MAH/g, were used in the adsorption/desorption of human serum albumin (HSA) from both aqueous solutions and human serum. The adsorption of HSA onto p(EGDMA–HEMA–MAH) was low (8.8 mg/g). Cu(II) chelation onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA adsorption was observed at pH 8.0 Higher HSA adsorption was observed from human serum (94.6 mg HSA/g). Adsorptions of other serum proteins were obtained as 3.7 mg/g for fibrinogen and 8.5 mg/g for γ‐globulin. The total protein adsorption was determined as 107.1 mg/g. Desorption of HSA was obtained using a 0.1M Tris/HCI buffer containing 0.5M NaSCN. High desorption ratios (to 98% of the adsorbed HSA) were observed. It was possible to reuse Cu(II)‐chelated p(EGDMA–HEMA–MAH) beads without significant decreases in the adsorption capacities. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 2840–2847, 2003