Characterization of the interaction between human serum albumin and morin

The interaction of morin with human serum albumin (HSA) has been investigated by using fluorescence, UV absorption and Fourier transform infrared spectroscopic approaches for the first time. Fluorescence data revealed the presence of a specific binding site on HSA for morin, and the binding affinity was 1.13+/-0.11x10(-5) L Mol(-1) in the physiological condition. The intrinsic fluorescence of morin was conspicuously enhanced in the presence of HSA due to excited-state proton transfer. The binding ability of morin to protein decreased with the increase of the buffer pH from 6.4 to 8.4, which signified that the level of protonation of the hydroxyl groups played an important role during the drug-protein binding process. From the UV absorption spectra of morin in various pH medium, the dissociation behaviors of the hydroxyl groups on the drug molecule were assigned. The second derivative UV absorption spectra of morin after interacting with HSA were used to elucidate the binding mode of morin to protein. The obvious red shift of the UV absorption band I of morin upon binding to HSA further confirmed the formation of HSA-morin complex, and this property was also utilized to estimate the binding constant. The interaction between morin and HSA induced an obvious reduction of the protein alpha-helix and beta-sheet structures.     

Stabilization mechanism of prostacyclin by human serum: an approach by binding kinetics using a stable prostaglandin I2 analogue, iloprost

We used a gel filtration method and a stable prostaglandin I2 (prostacyclin) analogue, iloprost, to study the kinetics of prostaglandin I2 binding by human serum proteins. Binding equilibrium experiments conducted at physiological prostaglandin I2 concentration (nM) yielded a KD of 10(-9) and a capacity of approx. 50 nM for the serum binding protein(s). Kinetic measurements gave a dissociation rate constant of 10(-3) s-1. When binding equilibrium was established at various ligand concentrations ranging from nM to microM, a result indicating an unsaturable binding was obtained utilizing this method. On the other hand, saturation was achieved with a ligand concentration as high as 50-100 microM by another binding method. A KD of 7 X 10(-5) and a capacity of approx. 600 microM was obtained. This apparent discrepancy was resolved by performing parallel experiments using purified human serum albumin samples and serum. It is concluded that the large quantity of serum albumin, approx. 600 microM, in serum may compensate for its low KD (approx. 10(-5] for prostaglandin I2, thus simulating a binding protein with a KD of 10(-9) and a limited capacity. These data offer direct information regarding how prostaglandin I2 is stabilized by serum and is transported to the platelet prostaglandin I2 receptors. There is a strong implication that serum albumin is the major if not the only protein responsible for binding of prostaglandin I2.     

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.     

The interaction between cholesterol and human serum albumin

The interaction between cholesterol and Human Serum Albumin (HSA) was studied by fluorescence technique. Addition of cholesterol causes decreasing of the fluorescence intensity of HSA and the mechanism can be attributed to static quenching. Both negative enthalpy and entropy change indicate this binding was an "enthalpy-driven" reaction. The number of binding site and distance between residues and ligands were also calculated: n = 0.98, r = 3.84 nm. UV-vis spectra showed HSA molecules unfolded to some extent and the hydrophobicity was decreased in the presence of cholesterol.     

Reactions of 2,6-dinitro-4-trifluoromethylbenzenesulfonate with human serum albumin

The reaction of the title compound with human serum albumin has been examined at various concentrations of the sulfonate. Kinetic data suggest that there are two highly reactive lysine amino groups on the protein, five lysine residues which are less reactive and an undetermined number of additional nucleophilic groups that react very slowly with the reagent at pH 7.5. One of the rapidly reacting lysines is tentatively identified as lysine-199 in the protein sequence. Fluorine NMR experiments indicate the presence of tight binding sites on the protein for the sulfonate which are not near reactive functional groups.     

Characterization of the interaction between nitrofurazone and human serum albumin by spectroscopic and molecular modeling methods

The interaction of nitrofurazone (NF) and human serum albumin (HSA) has been studied by fluorescence spectroscopy, FT-IR spectroscopy and molecular modeling methods. The results showed that the fluorescence of HSA was quenched by NF in a static quenching mechanism. Thermodynamic parameters revealed that hydrogen bonds and van der Waals force played the major role during the interaction. The calculated binding distance (r) indicated that the non-radioactive energy transfer came into being in the interaction between NF and HSA. HSA had a single class of binding site at Sudlow' site I in subdomain IIA for NF, which was verified by the displacement experiment. The molecular modeling study further confirmed the specific binding sites of NF on HSA, such as the interaction between N11 and N14 of NF with Lue 283 and Ser 287 predominately through hydrogen bonds. Three-dimensional fluorescence spectra indicated that the polarity around the tryptophan residues decreased and the conformation of HSA changed after adding NF. FT-IR spectra showed that NF could induce the polypeptides of HSA unfolding because it changed α-helix and β-sheet into β-turn and random structure of HSA.     

Exploring the interaction between tyrphostin 9 and human serum albumin using biophysical and computational methods

Abstract

Tyrphostin 9 (Tyr 9) is a potent platelet-derived growth factor receptor (PDGFR) inhibitor, which induces apoptosis in various cancer cell types. The binding of Tyr 9 to the major transport protein, human serum albumin (HSA) was investigated using several spectroscopic techniques and molecular docking method. Fluorescence quenching titration results showed progressive decrease in the protein fluorescence with increasing drug concentrations. A decreasing trend of the Stern-Volmer constant, K _sv with increasing temperature characterized the drug-induced quenching as static quenching, thus pointed towards the formation of Tyr 9–HSA complex. The binding constant of Tyr 9–HSA interaction was found to lie within the range 3.48–1.69?×?10⁵ M^?1 at three different temperatures, i.e. 15 °C, 25 °C and 35?°C, respectively and suggested intermediate binding affinity between Tyr 9 and HSA. The drug–HSA complex seems to be stabilized by hydrophobic forces, van der Waals forces and hydrogen bonds, as suggested from the thermodynamic data as well as molecular docking results. The far-UV and the near-UV CD spectral results showed slight alteration in the secondary and tertiary structures, respectively, of the protein upon Tyr 9 binding. Interaction of Tyr 9 with HSA also produced microenvironmental perturbations around protein fluorophores, as evident from the three-dimensional fluorescence spectral results but increased protein’s thermal stability. Both competitive drug binding results and molecular docking analysis suggested Sudlow’s Site I of HSA as the preferred Tyr 9 binding site.

Communicated by Ramaswamy H. Sarma     

Study on the interaction between human serum albumin and a novel bioactive acridine derivative using optical spectroscopy

The interaction of a novel bioactive agent N‐{[N‐(2‐dimethylamino) ethyl] acridine‐4‐carboxamide}‐α‐alanine [N‐(ACR‐4‐CA)‐α‐ALA] with human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption and circular dichroism spectrophotometric techniques under simulative physiological conditions. The fluorescence quenching of HSA by addition of N‐(ACR‐4‐CA)‐α‐ALA is due to static quenching and hydrogen bonding. Moreover, hydrophobic interactions play a role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA as well. The number of binding sites, n, and the binding constant values, K_A, were noted to be 0.88 and 3.4 × 10⁴ L mol^?1 for N‐(ACR‐4‐CA)‐α‐ALA at 293 K. The binding distances and the energy transfer efficiency between N‐(ACR‐4‐CA)‐α‐ALA and protein were determined. The negative value of enthalpy change and positive value of entropy change in the present study indicated that both hydrogen bonding and hydrophobic forces played a major role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA. Copyright © 2010 John Wiley & Sons, Ltd.     

Studies on the interaction between Ag(+) and human serum albumin

The interaction between Ag(+) and human serum albumin (HSA) has been intensively studied by means of equilibrium dialysis, ligand-to-metal charge transition (LMCT) bands, circular dichroism (CD) and Raman spectroscopy. Scatchard analysis of the results of equilibrium dialysis indicates the presence of two types of binding sites for Ag(+) on HSA, and the orders of magnitude of binding stability constants are found to be 10(5) and 10(4), respectively. During the binding process, a gradual increase in absorbance values of LMCT bands is observed with time-scanning UV absorption spectra, implying the Ag(I) centers are continually formed in HSA. The time-scanning CD spectra provide evidence that the binding of Ag(+) induces HSA to undergo a slow rearrangement of tertiary structure, and to change from the original conformation in the absence of Ag(+) (B-state) to conformation binding with Ag(+) (A-state). The rate constants and activation free energy of A-B transition are calculated. The Raman spectrum of Ag(I)-HSA system shows distinct vibration bands at 224 and 246 cm(-1) in the low-frequency region, which significantly reveal the formation of Ag-S and Ag-N bonds. In addition, the electrostatic interaction between Ag(+) and negatively charged oxygen is also detected with Raman spectroscopy.     

Characterization of the interaction between reserpine and bovine serum albumin: spectroscopic approaches

The characteristics of the interaction between reserpine and bovine serum albumin (BSA) were studied by fluorescence, UV-vis absorption and Fourier transform infrared (FT-IR) spectroscopy. Spectroscopic analysis revealed that fluorescence quenching of BSA by reserpine was through a static quenching procedure. The binding constant K(A) of reserpine with BSA at 293, 301 and 309 K was 1.63, 1.78 and 2.35 x 10(5) moL(-1) L respectively, which indicated degree of binding force between reserpine and BSA. There was one binding site between reserpine and BSA. The entropy and enthalpy changes were positive, indicating that interaction of reserpine and BSA was driven mainly by hydrophobic forces. The average binding distance between the donor (BSA) and the acceptor (reserpine) was about 3.84 nm based on the Forster non-radiation energy transfer theory. Results of synchronous fluorescence and FT-IR spectra indicated that the conformation and microenvironment of BSA were changed by the binding of reserpine. The results may provide important insights into the physiological activity of reserpine.     

Study of the interaction between fisetin and human serum albumin: a biophysical approach

The binding of fisetin with human serum albumin (HSA) has been studied at different pH using UV-Vis, FTIR, CD and fluorescence spectroscopic techniques. The binding constants were found to increase with the rise in pH of the media. The negative ΔH° (kJ mol-1) and positive ΔS° (J mol-1 K-1) indicate that fisetin binds to HSA via electrostatic interactions with an initial hydrophobic association that result in a positive ΔS° . In presence of potassium chloride (KCl) the binding constants were found to be decrease. The α-helical content of HSA increased after binding with fisetin as analyzed from both CD and FTIR methods. The site marker displacement studies using fluorescence anisotropy suggest that fisetin binds to the hydrophobic pocket (Site 1, subdomain IIA) of HSA which is in good accordance with the molecular docking study. The change in accessible surface area (ASA) of residues of HSA was calculated to get a better insight into the binding.     

Spectroscopic studies of the interaction between hypocrellin B and human serum albumin

Previous work has proved that hypocrellin B (HB) binds to human serum albumin (HSA) at a specific site instead of distributed randomly on the surface of a protein. In the current work, further investigation by using bilirubin as a site I marker indicates that HB can compete for the same site with bilirubin, suggesting that the HB binding site is located at sub-domain IIA (site I) of HSA. Moreover, bound to HSA, the HB fluorescence was found to be pH sensitive in physiological range (pH 6.0-8.0). The increasing of binding constant of HB to HSA in the pH range 6-8 also indicates that the N<-->B transition modulates the microenvironment changes of the binding site and influences considerably the binding between HB and HSA. Furthermore, picosecond time-resolved fluorescence spectra of HB-HSA complex in PBS indicate an additional short-lived component compared to that for HB in benzene, which may be assigned to the process of electron transfer from Trp-214 to HB.     

Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach

The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT–IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were ?4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non‐radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na⁺, K⁺, Li⁺, Ni²⁺, Ca²⁺, Zn²⁺ and Al³⁺ were found to influence binding of the drug to protein. The 3D fluorescence, FT–IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Study of the interaction between ofloxacin and human serum albumin by spectroscopic methods

The binding of ofloxacin (OFLX) to human serum albumin (HSA) was investigated by fluorescence and circular dichroism (CD) techniques. The binding parameters have been evaluated by a fluorescence quenching method. Competitive binding measurements were performed in the presence of warfarin and ibuprofen and suggest binding to the warfarin site I of HSA. The distance r between donor (HSA) and acceptor (OFLX) was estimated according to the Forster's theory of non‐radiatiative energy transfer. CD spectra revealed that the binding of OFLX to HSA induced conformational changes in HSA. Molecular docking was performed and shows that for the lowest energy complex OFLX is located in site I of HSA, which correlate to the competitive binding experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparative investigation on interaction between potent antimalarials and human serum albumin using multispectroscopic and computational approaches

This study performed a comparative investigation to explore the interaction mechanisms between two potential antimalarial compounds, JMI 346 and JMI 105, and human serum albumin (HSA), a vital carrier protein responsible for maintaining important biological functions. Our aim was to assess the pharmacological efficiency of these compounds while comprehensively analyzing their impact on the dynamic behavior and overall stability of the protein. A comprehensive array of multispectroscopic techniques, including UV–Vis. spectroscopy, steady-state fluorescence analysis, synchronous fluorescence spectroscopy, three-dimensional fluorescence and circular dichroism spectroscopy, docking studies, and molecular dynamics simulations, were performed to probe the intricate details of the interaction between the compounds and HSA. Our results revealed that both JMI 346 and JMI 105 exhibited promising pharmacological effectiveness within the context of malaria therapy. However, JMI 346 was found to exhibit a significantly higher affinity and only minor altered impact on HSA, suggesting a more favorable interaction with the protein on the dynamic behavior and overall stability of the protein in comparison to JMI 105. Further studies can build on these results to optimize the drug–protein interaction and enable the development of more potent and targeted antimalarial treatments.     

A study on the allosteric interaction between the major binding sites of human serum albumin using microcalorimetry

The binding of warfarin and oxyphenbutazone to albumin has been studied at pH 6.8 and pH 9.2 by measuring the heat of binding of these ligands to their high-affinity binding sites on albumin (delta Ho'1). The -delta Ho'1 values for the binding of warfarin at pH 6.8 and 9.2 and oxyphenbutazone at pH 6.8 and 9.2 were found to be 16.9(+/- 0.6), 28.8(+/- 0.6), 10.5(+/- 0.4) and 17.4(+/- 0.6) kJmol-1, respectively. The Gibbs energies (delta Go'1) corresponding to these delta Ho'1 values cover a much smaller range. The pH dependences of delta Go'1 and delta Ho'1 are explained in terms of pK shifts in the albumin upon binding warfarin or oxyphenbutazone. Diazepam, which binds to a site on albumin which is different from the warfarin-oxyphenbutazone binding site, increases - delta Ho'1 for the binding of warfarin and oxyphenbutazone to albumin at pH 6.8, but it does not influence the -delta Ho'1 at pH 9.2. This phenomenon may be attributed to an allosteric interaction between the diazepam binding site and the warfarin binding site. This allosteric interaction must have its origin in a phenomenon other than the N-B transition.     

RNA-hydrolyzing activity of human serum albumin and its recombinant analogue

The comparative analysis of RNA-hydrolyzing activity of albumin from human serum and albumin expressed in methylotrophic yeast Pichia pastoris has been carried out. The rate of polyribonucleotide phosphodiester bond cleavage in the presence of recombinant albumin has been found to be similar to that of the reaction mediated by the native protein. According to ³¹P NMR data, RNA hydrolysis follows the mechanism of intermolecular trans-esterification to yield 2′,3′-cyclophosphodiester reaction products that are further slowly hydrolyzed to form nucleoside-3′- and nucleoside-2′-phosphates. Analysis of pH dependence suggests an acid–base mechanism of catalysis. The catalytic activity and substrate specificity of albumin in RNA hydrolysis distinguish it from human ribonucleases.     

A prostacyclin analogue, iloprost, augments the ovulatory response of the LH-stimulated in vitro perfused rat ovary

Ovaries from immature rats, primed with pregnant mare's serum gonadotropin (PMSG; 20 IU, on day 28), were perfused in vitro in a recirculating system for 21 h from the morning of day 30 of age. Stimulation with luteinzing hormone (LH; 0.1 μg/ml) in vitro at 0 h of perfusion resulted in 2.4 ± 0.75 (mean ± SEM) ovulatioons per treated ovary, whereas no ovulations occured in the unstimulated group. When the addition of LH was supplemented hourly for 10 h with a stable prostacyclin analogue, Iloprost, at concentrations of 0.01 μM or 0.1 μM, the ovulation rate increase significantly (p<0.05) to 6.6 ± 1.3 and 10.2 ± 2.4 ovulations per treated ovary, respectively. Iloprost (0.1 μM) did not cause any follicular ruptures when added by itself at every hour up to 10 h. The addition of Iloprost did not affect the release of cyclic adenosine 3′,5′-monophosphate or LH-stimulated ovaries. All ovulated oocytes had resumed meiosis as judged from the absence of a germinal vesicle. These data indicate a positve modulatory role of prostacyclin in the LH-induced ovulatory process for the rat.     

Kinetics and mechanism of the interaction between human serum albumin and monomeric haemin.

The interaction of human serum albumin with monomeric haemin has been investigated by detailed kinetic analysis in dimethyl sulphoxide/water (3:5, v/v). The results obtained under conditions of albumin saturation of haemin and under pseudo-single turnover conditions indicate that methaemalbumin is formed in a two-stage, single-intermediate process. The initial association between the haemin and human serum albumin is a chemically controlled process (k1 = 1.7 X 10(5) mol-1 . s-1 . dm3 at 24 degrees C); the variation of K1 with pH exhibited a well defined pK of 5.9. The overall equilibrium constant, calculated by using microscopic rate constants, is 1.1 (+/- 0.5) X 10(8) mol-1 at 24 degrees C. The data and conclusions are consistent with a general binding mechanism for albumin in which intermediate formation is followed by an entropy-controlled internalization of the ligand.