The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.     

The comparison of biophysical properties of DB-67 and its ester DB-67-4ABTFA determined by fluorescence spectroscopy methods

Fluorescence spectroscopy methods are applied to the study of camptothecin analogue DB-67 and its ester DB-67-4ABTFA (trifluoroacetic acid salt of 20(S)-aminobutyrate substituted DB-67). Camptothecin and many of its analogues exhibit anticancer properties. They are fluorescent compounds, so using the method of fluorescence anisotropy measurements and fluorescence spectra recording many biophysical properties can be determined including affinity to proteins and membranes. One can also observe the process of conversion of the ester into DB-67. Active lactone form of camptothecin in fluids at pH 7.4 hydrolyses and converts into inactive carboxylate. Process of camptothecin deactivation is accelerated in plasma and after about 2h the total conversion to carboxylate form occurs. It is caused by fast and irreversible binding of carboxylate form to the human serum albumin (HSA). Camptothecin carboxylate bound to HSA does not lactonise. On the other hand, camptothecin lactone binding to membranes is reversible, but as long as lactone form bound to membranes does not hydrolyse. Knowledge of binding properties to proteins and membranes permits to select among many camptothecin analogues the ones exhibiting desirable behavior in physiological conditions: high affinity of lactone form to membranes and low affinity of carboxylate form to albumin. The studied DB-67 and DB-67-4ABTFA fulfill these requirements.     

Study of characterization and application on the binding between 5-iodouridine with HSA by spectroscopic and modeling

The binding of 5-iodouridine with human serum albumin was investigated under the simulative physiological conditions. The fluorescence spectra in combination with UV absorption and modeling method were used in the present work. A strong fluorescence quenching reaction of 5-iodouridine to HSA was observed and the quenching mechanism was suggested as static quenching procedure. The binding constants (K) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), were calculated. It showed that the hydrophobic interaction was a predominant intermolecular force in order to stabilize the complex, which was in agreement with the result of modeling study. The binding distance between 5-iodouridine and HSA was calculated on the basis of the theory of Föster energy transfer. The effects of other ions on the binding constants were also discussed. Synchronous fluorescence spectroscopy (SFS) technique were successfully applied to determine protein in the biological samples.     

Effects of homogeneous media, binary mixtures and microheterogeneous media on the fluorescence and fluorescence probe properties of some benzo[b][1,8]naphthyridiens with HSA and BSA

A rapid and efficient method for the synthesis of various poly‐substituted benzo[b][1,8]naphthyridines in high yield has been developed via the Friedländer condensation of 2‐aminoquinoline‐3‐carbaldehyde 1 with various alicyclic ketones in a base catalyst (aq. potassium hydroxide). A series of benzo[b][1,8]naphthyridines branched with various side‐chains and substituents were prepared with the aim of being investigated as a fluorescent agents. Electronic absorption and fluorescence properties of some representative benzonaphthyridines (3d, 5b and 21f) in homogeneous organic solvents, dioxane–water binary mixtures and in the microheterogeneous media (sodium dodecyl sulphate (SDS), cetyl trimethyl ammonium bromide (CTAB) and Triton‐X100 micelles) have been examined. A linear correlation between solvent polarity and fluorescence properties was observed. Further, the interaction of these benzonaphthyridines (3d, 5b and 21f) with human serum albumin (HSA) and bovine serum albumin (BSA) in phosphate buffer have been examined by UV‐vis absorption and fluorescence spectroscopy. The fluorescence intensity of 3d, 5b and 21f increases with the increasing HSA and BSA concentration. These benzonaphthyridines also quench the 345 nm fluorescence of BSA in phosphate buffer (λ_ex 280 nm). These compounds have potential for use as neutral and hydrophobic fluorescence probes for examining the microenvironments in proteins, polymers, micelles, etc. Copyright © 2011 John Wiley & Sons, Ltd.     

Investigations of the effects of ethanol on warfarin binding to human serum albumin

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.     

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site, proved to be the preferential one, being 6.50 x l0(5), 1.94 x 10(6) and 8.94 x 10(5), respectively. In addition, it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions.     

The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study

Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient F?rster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.     

Polarographic direct determination and homogeneous immunoassay of anti-human serum albumin (HSA) by parallel catalytic hydrogen wave of anti-HSA or HSA

Human serum albumin (HSA) or anti-human serum albumin (anti-HSA) yields a catalytic hydrogen wave at about -1.85V (vs Ag/AgCl) in 0.25M NH(3).H(2)O-NH(4)Cl (pH 8.58) buffer. When 1.0 x 10(-2)M K(2)S(2)O(8) is present, the catalytic hydrogen wave is further catalyzed, producing a parallel catalytic wave of hydrogen as catalyst in nature, termed the parallel catalytic hydrogen wave. The sensitivity of the parallel catalytic hydrogen wave is higher by two orders of magnitude than that of the catalytic hydrogen wave. Using the parallel catalytic hydrogen wave of anti-HSA or HSA in the presence of K(2)S(2)O(8), two sensitive methods for the determination of anti-HSA were developed. One is a direct determination based on the parallel catalytic hydrogen wave of anti-HAS itself, and the other is a homogeneous immunoassay based on measuring the decrease of the peak current of the parallel catalytic hydrogen wave of HSA after homogeneous immunoreaction of HSA with anti-HSA. In the direct determination, the second-order derivative peak current of the parallel catalytic hydrogen wave of anti-HSA itself is rectilinear to its titer in the range from 1:1.0 x 10(7) to 1:8.4 x 10(6). In the homogeneous immunoassay, the decrease in the second-order derivative peak current of the parallel catalytic hydrogen wave of HSA is linearly related to the added anti-HSA in the titer range from 1:3.0 x 10(7) to 1:6.0 x 10(6). These assays are highly sensitive and rapid in operation and can be used to evaluate such antigens and their antibodies as those that could yield the parallel catalytic hydrogen wave.     

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.     

新型人血清白蛋白特异结合肽ML的筛选及鉴定

利用噬菌体展示技术筛选和鉴定新型人血清白蛋白(HSA)特异结合7肽,分析与垂体腺苷酸环化酶激活肽27(PACAP27)构成的融合多肽ML-PACAP27与HSA的亲和结合力,以确定筛选的7肽(ML1和ML2)在与其他药物多肽或蛋白融合状态下仍具有较高的HSA结合活力。利用噬菌体展示7肽库,经四轮筛选、筛选克隆的DNA测序、酶联免疫吸附技术分析(ELISA)初步鉴定筛选克隆与HSA的亲和作用,并利用等离子共振技术(SPR)定量测定合成的ML-PACAP27融合多肽与人血清白蛋白的亲和力常数。实验结果表明:经筛选获得2个与人血清白蛋白特异性结合的7肽序列,其氨基酸序列分别为:ML1:LKSCKPL和ML2:SLKSHAL;ELISA分析结果显示,含有ML1和ML2的噬菌体均可亲和结合HSA,而且ML2的亲和结合作用高于ML1;SPR分析结果显示,ML1-PACAP27和ML2-PACAP27与HSA的解离常数(KD)分别为:8.1×10-6mol/L和3.7×10-6mol/L,ML1-PACAP27与HSA的结合力高于ML1-PACAP27。筛选和鉴定了两个可与HSA特异性结合的7肽序列,该7肽序列可用于特异偶联HSA的长效分子药物的重组融合表达或设计,可为延长分子药物的半衰期及新型药物研发提供新工具。     

蛋白质与邻二氮菲相互作用的单扫描极谱法研究

在pH 4.7的HAc-NaAc缓冲溶液中,邻二氮菲与蛋白质相互作用,使邻二氮菲在-0.99 V (vs.SCE)处的还原峰电流下降,电流降低值与所加的蛋白质(人血清白蛋白、牛血清白蛋白、溶菌酶)的量在一定范围内呈线性关系,线性范围分别为2.0～22,2.0～20,4.0～26 mg/L;检测限分别为1.0,1.0,2.0 mg/L。应用该方法测定了人血清中白蛋白的含量,其结果令人满意。     

Characterization of the interaction between 2′-deoxyuridine and human serum albumin

The binding of 2′-deoxyuridine to human serum albumin (HSA) was investigated by fluorescence spectroscopy in combination with molecular modeling under simulation of physiological conditions. The quenching mechanism was suggested to be static according to the fluorescence measurement. The thermodynamic parameters: enthalpy change (ΔH) and entropy change (ΔS) were calculated to be −18.87 kJ/mol and 24.00 J/(mol K) according to the Vant’Hoff equation. These data suggest that hydrophobic interactions are the predominant intermolecular forces stabilizing the complex. Experimental results are in agreement with the results obtained by molecular modeling study. In addition, the effects of common ions on the binding constants were also studied at room temperature.     

Characterization of the binding of sulfonylurea drugs to HSA by high-performance affinity chromatography

Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (K_a) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average K_a values of 1.3 (±0.2) × 10⁵ and 3.5 (±3.0) × 10² M⁻¹ for acetohexamide and values of 8.7 (±0.6) × 10⁴ and 8.1 (±1.7) × 10³ M⁻¹ for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with K_a values of 1.3 (±0.1) × 10⁵ and 4.3 (±0.3) × 10⁴ M⁻¹, respectively, at 37 °C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with K_a values of 5.5 (±0.2) × 10⁴ and 5.3 (±0.2) × 10⁴ M⁻¹, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug–protein interactions.     

The fluorescence and circular dichroism of proteins in reverse micelles: application to the photophysics of human serum albumin and N-acetyl-l-tryptophanamide

Evidence is presented that a compartmentalised protein exists in its native state only within a particular size of aqueous cavity. This behaviour is shown to exist in AOT reverse micelles using fluorescence quenching and circular dichroism (CD) studies of human serum albumin (HSA). In particular, far ultraviolet CD measurements show that a reduction in quencher accessibility to the fluorophore is consistent with the protein being nearest to its native conformation at a waterpool size of around 80 Å diameter. We also show that the biexponential fluorescence decay of N-acetyl-l-tryptophanamide (NATA) in AOT reverse micelles arises from the probe being located in two distinct sites within the interfacial region. The more viscous of these two sites is located on the waterpool side of the interface and the other is located on the oil side of the interface.     

Study on the interaction between DNA and protein induced by anticancer drug carboplatin

The interaction of DNA and human serum albumin (HSA) in the presence of anticancer drug carboplatin was studied with piezoelectric quartz crystal impedance (PQCI) and electrochemistry techniques. In the PQCI analysis, the correlative parameters including the frequency (f0), the motional resistance (R(m)), and the static capacitance (C0) in the experiment were obtained and discussed in detail. Additionally, the kinetics parameters of the cross-linking process were investigated and a response kinetics model was deduced. The values of association rate constant k1, dissociation rate constant k(-1) and the reaction equilibrium constant K were estimated to be 1.895 mg/ml(-1) s(-1), 4.7 x 10(-5) s(-1) and 4.03 x 10(4) (mg/ml)-1, respectively. Furthermore, cyclic voltammetry (CV) and electrochemical AC impedance techniques were employed to testify the cross-linking process.     

High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK

The bisubstrate fluorescent probe ARC-583 (Adc-Ahx-(d-Arg)₆-d-Lys(5-TAMRA)-NH₂) and its application for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of protein kinases PKA (cyclic AMP-dependent protein kinase) and ROCK (rho kinase) in fluorescence polarization-based assay are described. High affinity of the probe (K_D = 0.48 nM toward PKA) enables its application for the characterization of inhibitors with nanomolar and micromolar potency and determination of the active concentration of the kinase in individual experiments as well as in the high-throughput screening format. The probe can be used for the assessment of protein-protein interactions (e.g., between regulatory and catalytic subunits of PKA) and as a cyclic AMP biosensor.     

Coumarin tethered cyclic imides as efficacious glucose uptake agents and investigation of hit candidate to probe its binding mechanism with human serum albumin

A series of novel coumarin-cyclic imide conjugates (1a–1j) were designed and synthesized to evaluate their glucose uptake activity by insulin resistant liver hepatocyte carcinoma (HepG2) cells through 2-NBDG uptake assay. Compounds (1a–1j) were characterised using various analytical methods such as ¹H NMR, ¹³C NMR, IR, GC–MS, elemental and single-crystal X-ray diffraction techniques. Compounds (1a–1j) exhibited 85.21 – 65.80% of glucose uptake and showed low level of cytotoxicity towards human embryonic kidney cells (HEK-293) indicating good selectivity and safety profile. Compound 1f was identified as a hit candidate exhibiting 85.21% of glucose uptake which was comparable with standard antidiabetic drug Metformin (93.25% glucose uptake). Solution stability study under physiological pH conditions ≈ (3.4 – 8.7), indicates that compound 1f is sufficiently stable at varied pH conditions and thereby compatible with bio-physiological environments. Interaction of 1f with human serum albumin (HSA) were also studied which quantifies that compound 1f binds with HSA efficiently through facile binding reaction in solution. Fluorescence, UV–vis spectrophotometry and molecular modeling methodologies were employed for studying the interaction mechanism of compound 1f with protein.     

Investigation of interaction between human serum albumin and N6-(2-hydroxyethyl)-adenosine by fluorescence spectroscopy and molecular modelling.

The interaction between human serum albumin (HSA) and N(6)-(2-hydroxyethyl)-adenosine (HEA) was investigated using fluorescence spectroscopy in combination with UV absorption spectroscopy for the first time. The results of spectroscopic measurements suggested that the hydrophobic interaction was the predominant intermolecular force stabilizing the complex, which was in good agreement with the results of molecular modelling study. The enthalpy change (DeltaH) and the entropy change (DeltaS) were calculated, according to the Van't Hoff equation, to be -24.05 kJ/mol and 30.23 J/mol/K, respectively. The effects of common ions on the binding constant of the HEA-HSA complex at room temperature were also investigated.     

Time-dependent decay and anisotropy of fluorescence from diphenylhexatriene embedded in the chloroplast thylakoid membrane

The hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene has been incorporated into the membranes of isolated thylakoids, separated granal and stromal lamellae and aqueous dispersions of extracted thylakoid galactolipids. Time-resolved fluorescence decays have been recorded on a nanosecond scale using single-photon counting in order to assess the motional properties of the probe. All the experimental systems used showed biphasic decay kinetics and the anisotropies of the decays have been interpreted in terms of a model for wobbling diffusion confined to a cone. The analysis has given information about dynamic and structural restraints of the lipid acyl chains. In the intact thylakoid membrane the degree of order of the fatty acid acyl chains is higher and their rate of motion slower than for isolated lipids. Even so, the dynamic and structural parameters indicate that the thylakoids can be considered as a relatively fluid membrane system when compared with many other biological membranes, a property which is probably required to facilitate efficient long-range diffusion of lipophilic mobile electron-transport components. It is suggested that the optimization of thylakoid fluidity is linked to regulation of the membrane protein/lipid ratio which is also likely to be responsible for the higher fluidity of stromal membranes relative to those of the grana.     

Binding of all-trans retinoic acid to human serum albumin: fluorescence, FT-IR and circular dichroism studies

All-trans retinoic acid derived from vitamin A is an essential component for the modulation of angiogenesis, the process of blood vessel formation. We have investigated the binding of all-trans retinoic acid to the carrier protein, human serum albumin (HSA) under physiological conditions. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy were used for the biophysical studies. The binding parameters were determined by a Scatchard plot and the results found to be consistent with those obtained from a modified Stern–Volmer equation. From the thermodynamic parameters calculated according to the van’t Hoff equation, the enthalpy change ΔH⁰ and entropy change ΔS⁰ are found to be 106.17 and 106.14 J/mol K, respectively. These values suggest that apart from hydrophobic interactions electrostatic interactions are present. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. Docking studies performed substantiated our experimental findings and it was observed that all-trans retinoic acid hydrogen bonded with Trp 214 and Asp 451 residues of subdomain IIA and IIIA of HSA, respectively.