HSA Complex Research Articles

Synthesis, characterization and interaction of N,N′-dipyridoxyl (1,4-butanediamine) Co(III) salen complex with DNA and HSA

Co(III) salen complex with N,N′-dipyridoxyl (1,4-butanediamine) Schiff-base ligand as tetradentate ligand was synthesized and characterized by the elemental and spectroscopic analysis. The interaction of this complex with calf thymus DNA (ct DNA) has been investigated in vitro using UV absorption, fluorescence spectroscopy, thermal denaturation and gel electrophoresis techniques. The binding constant has been estimated to be 1×104M−1 using UV absorption. The addition of ct DNA to Co(III) salen solution resulted in a fluorescence quenching. The binding constant and site size binding have been calculated in connection with other experimental observations show that the interactive model between Co(III) salen and ct DNA is an intercalative one. The interaction between plasmid DNA (pTZ57R DNA) and this complex is confirmed by gel electrophoresis studies. Furthermore, the interaction between HSA and Co(III) salen complex was investigated by UV absorption, fluorescence spectroscopy and molecular modeling. The binding constant for the interaction of this complex with HSA were found to be 3.854×104M−1 using UV absorption, which was in good agreement with the binding constant obtained from fluorescence method (3.866×104M−1). The binding distance between HSA and this complex was estimated to be 2.48nm according to Förster theory of non-radioactive energy transfer. Molecular modeling studies suggested that hydrophobic interaction was the predominant intermolecular forces stabilizing Co(III) complex-HSA system.

Investigation the interaction of Daphnin with human serum albumin using optical spectroscopy and molecular modeling methods

The interaction between Daphnin with human serum albumin has been studied for the first time by spectroscopic methods including fluorescence quenching technology, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that Daphnin can quench the intrinsic fluorescence of HSA by static quenching and there is a single class of binding site on HSA. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the protein secondary structure changed with increases of α-helices at the drug to protein molar ratio of 2. Furthermore, the thermodynamic functions ΔH0 and ΔS0 for the reaction were calculated to be 11.626kJmol−1 and 118.843Jmol−1K−1 according to Van’t Hoff equation. The thermodynamic parameters (ΔH0 and ΔS0) and the molecular modeling study indicated that hydrophobic force played an important role to stabilize the Daphnin–HSA complex, and Daphnin could bind within the subdomain IIA of the HSA.

Locating the binding sites of Pb(II) ion with human and bovine serum albumins.

Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization.

Relative Affinities of Fatty Acid Binding Sites on Human Serum Albumin Probed by 2D-NMR

In circulation HSA is the principal carrier for endogenous lipophilic compounds, primarily non-esterified long chain fatty acids (FA). Since FA bind to multiple binding sites with varying affinities, it would be useful to probe the relative affinities for these FA binding sites with a method that distinguishes individual binding sites. As visualized in crystal structures FA binding distributes asymmetrically throughout the protein. In the physiologically relevant solution state, these sites were partially distinguished by 1D-13C-NMR spectroscopy and subsequently correlated with crystal structure sites. Here we show nine, well-resolved peaks in 1H-13C-NMR spectra of 18-13C-oleic acid (OA) / HSA complexes. Different NMR signals represent FA bound at different sites throughout the protein, with the varying intensities corresponding to the different affinities HSA has for OA. This investigation probes the relative affinities of FA with three approaches: (i) addition of different FA molar ratios to HSA, to observe the order of filling of binding sites; (ii) addition of FA acceptors to observe the dissociation of FA from HSA; and (iii) addition of drugs that are known to bind to low affinity FA sites. From the order of filling, the three highest affinity-binding sites are clearly differentiated from the six lower/medium affinity-binding sites at the physiologically relevant FA:HSA molar ratio- 4:1. Methyl-β-cyclodextrin (MβCD) extracted FA from individual sites, in a concentration dependent manner, with the highest concentrations removing FA from the highest affinity sites. Relative affinities determined as above were consistent with the binding of drugs to Sudlow's drug binding sites, which displaced bound FA from specific lower affinity sites. 2D-NMR spectroscopy is a powerful approach for studying interactions of FA with HSA and FA-competitive drugs in a site-specific manner, providing a unique view of HSA-ligand binding.

A Thermodynamic Investigation of Aspirin Interaction with Human Serum Albumin at 298 and 310K

This study was designed to determine the structural changes of human serum albumin, HAS, in the presence of aspirin in H 2 O solutions at physiological pH 7. Isothermal titration calorimetry, ITC, at 298 and 310K, and curve-fitting procedures were applied to characterize the drug binding sites, the binding constants in the aspirin+HSA complexes. The heats obtained for aspirin+HSA interactions are reported and analyzed in terms of the extended solvation theory. It was indicated that there are a set of two identical and non-cooperative sites for aspirin. Calorimetric evidence showed that strong aspirin+HSA interaction occurs at very low aspirin concentration (0.007-0.084 mM), with overall binding constants of 2.76 × 10 6 M -1 and 9.3×10 5 M -1 at low and high aspirin concentration domains respectively.

Determinations of Uranium(VI) Binding Properties with some Metalloproteins (Transferrin, Albumin, Metallothionein and Ferritin) by Fluorescence Quenching

The interactions between uranium and four metalloproteins (Apo-HTf, HSA, MT and Apo-EqSF) were investigated using fluorescence quenching measurements. The combined use of a microplate spectrofluorometer and logarithmic additions of uranium into protein solutions allowed us to define the fluorescence quenching over a wide range of [U]/[Pi] ratios (from 0.05 to 1150) at physiologically relevant conditions of pH. Results showed that fluorescence from the four metalloproteins was quenched by UO(2)(2+). Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of metalloproteins, i.e. binding constants and binding sites densities, were determined using classic fluorescence quenching methods and curve-fitting software (PROSECE). It was demonstrated that in our test conditions, the metalloprotein complexation by uranium could be simulated by two specific sites (L(1) and L(2)). Results showed that the U(VI)-Apo-HTf complexation constant values (log K(1)=7.7, log K(2)=4.6) were slightly higher than those observed for U(VI)-HSA complex (log K(1)=6.1, log K(2)=4.8), U(VI)-MT complex (log K(1)=6.5, log K(2)=5.6) and U(VI)-Apo-EqsF complex (log K(1)=5.3, log K(2)=3.9). PROSECE fitting studies also showed that the complexing capacities of each protein were different: 550 moles of U(VI) are complexed by Apo-EqSF while only 28, 10 and 5 moles of U(VI) are complexed by Apo-HTf, HSA and MT, respectively.

Affinity CE Determination of the Binding Constant of Bioactive Sulfated Polysaccharide 916 to Human Serum Albumin

Drug-protein binding is an important process in determining the activity of a pharmaceutical agent once it has entered the body. This paper developed an affinity capillary electrophoresis method to determine the binding constant between a bioactive sulfated polysaccharide 916 (916) and a potential protein, human serum albumin. This method is based on the principle that the changing analytes have different mobility shift in the zone electrophoresis. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant of 916 to the HSA complex. The binding constant Ka of 916 to the human serum albumin achieved was 2.1 × 104.

Study of the interaction between esculetin and human serum albumin by multi-spectroscopic method and molecular modeling

Esculetin derived from Cortex fraxin plays an important role as a traditional Chinese medicine because of its unique pharmacological properties. The interactions between esculetin and HSA were studied by fluorescence spectroscopic techniques under similar to human physiologic conditions. The binding parameters have been evaluated by fluorescence quenching methods. The results proved the mechanism of fluorescence quenching of HSA while interacting with esculetin is due to the formation of esculetin–HSA complex formation. The thermodynamic parameters like Δ H 0 and Δ S 0 were calculated to be −14.62 kJ/mol and 38.93 J/mol/K, respectively, which proves main interaction between esculetin and HSA is hydrophobic contact, but the electrostatic interaction cannot be excluded, which in agreement with the result of molecular docking study. The distance r between donor (HSA) and acceptor (esculetin) was obtained according to the Förster’s theory of non-radiative energy transfer and found to be 2.89 nm. From the high value of fluorescence anisotropy ( r = 0.07) it was argued that the probe molecular was located in motionally restricted environment of the protein. The alterations of protein secondary structure in the presence of esculetin were confirmed by the evidences from UV, FT-IR and CD spectroscopes. In addition, the effects of common ions and amino acids on the constants of esculetin–HSA complex were also discussed.

Crocetin, Dimethylcrocetin, and Safranal Bind Human Serum Albumin: Stability and Antioxidative Properties

Crocetin (CRT) and dimethylcrocetin (DMCRT) are derived from crocins which are found in the stigmas of saffron (Crocus sativus L.), while safranal is the main component of saffron's essential oil. The aim of the present study was to examine their interaction with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various ligand contents. FT-IR and UV-visible spectroscopic methods were used to determine the ligands' binding mode, the binding constant, and the effects of ligand complexation on protein secondary structure. Structural analysis showed that crocetin, dimethylcrocetin, and safranal bind nonspecifically (H-bonding) via protein polar groups with binding constants of Kcrt =2.05 (+/-0.30) x 103 M-1, Kdmcrt = 9.60 (+/-0.35) x 104 M-1, and Ksaf = 2.11 (+/-0.35) x 103 M-1. The protein secondary structure showed no major alterations at low ligand concentrations (1 microM), whereas at higher content (1 mM), decrease of alpha-helix from 55% (free HSA) to 43-45% and increase of beta-sheet from 17% (free HSA) to 18-22% and random coil 7% (free HSA) to 10-14% occurred in the ligand-HSA complexes. The results point to a partial unfolding of protein secondary structure at high ligand content. The antioxidant activity of CRT, DMCRT, and safranal was also tested by the DPPH* antioxidant activity assay, and their IC50 values were compared to that of well-known antioxidants such as Trolox and butylated hydroxy toluene (BHT). The IC50 values of CRT and safranal were 17.8 +/- 1 microg/mL and 95 +/- 1 microg/mL, respectively, while the inhibition of DMCRT reached a point of 38.8%, which corresponds to a concentration of 40 microg/mL, and then started to decrease. The IC50 values of Trolox and BHT were 5.2 +/- 1 microg/mL and 5.3 +/- 1 microg/mL, respectively.

Daidzein interaction with human serum albumin studied using optical spectroscopy and molecular modeling methods

In this work, fluorescence anisotropy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling methods were used to characterize optical properties of the Daidzein–HSA complex and to gain information on the binding mechanism at molecular level. Daidzein is weakly fluorescent in aqueous buffer medium, with an emission maximum at 466 nm. Binding of Daidzein with HSA leads to dramatic enhancement in the fluorescence intensity and anisotropy ( r), along with significant changes in the fluorescence excitation and emission profiles. The binding constant ( K = (5.9 ± 0.6) × 10 4 M −1) and the standard free energy change (Δ G ≈ −27.5 kJ/mol) for Daidzein–HSA interaction have been calculated from the relevant fluorescence data. The secondary structure compositions of free HSA and its Daidzein complexes were estimated by the FT-IR spectra and the curve-fitted results of amide I band, which are in good agreement with the analyses of CD spectra. Furthermore, the displacement experiments indicated that Daidzein can bind to the site I of HSA which is also in agreement with the result of molecule modeling study.

1H NMRD profiles and ESR lineshapes of Gd(III) complexes: a comparison between the generalized SBM and the stochastic Liouville approach

A complete description of the T 1-NMRD profiles and the ESR lineshape of Gd(III) complexes ( S = 7/2) was presented using second-order perturbation theory (GSBM) by Zhou et al. [J. Magn. Reson. 167 (2004) 147]. This report compares the GSBM with the stochastic Liouville approach (SLA) to determine the validity of the closed analytical expressions of NMRD and the ESR lineshape functions. Both approaches give the same results at high fields while a very small divergence is observed for X- and W-band ESR lineshapes when the magnitude of the perturbation term times the correlation time approaches the limit of the perturbation regime, Δ ZFS τ f ≈ 0.1. There was a clear discrepancy between the theoretical GSBM X-band spectrum and the recorded ESR spectrum of the Gd(III) MS-325 + HSA complex. This is probably due to a slow-motion effect caused by a slow modulation of the ZFS interaction. The characteristic correlation time of this slow modulation is in the range of 150 ps, which therefore cannot be due to the reorientational motion of the whole MS-325 + HSA complex.

Effects of labeling methods on the biodistribution of 99mTc-labeled HSA complexes

Effects of labeling methods on the biodistribution of 99mTc-labeled HSA complexes

Unique, pH-dependent biphasic band shape of the visible circular dichroism of curcumin–serum albumin complex

Interaction between the plant derived polyphenolic type curcumin molecule having anticarcinogenic, antiinflammatory, and antioxidant activities, and human serum albumin was studied at different pH values by circular dichroism (CD) and electronic absorption spectroscopy. The weak, induced CD spectrum of curcumin–HSA complex measured at pH 7.4 in the visible spectral region shows striking changes upon alkalization; CD spectra collected between pH 7.7 and 9.3 exhibit characteristic, oppositely signed CD band pair according to the visible absorption band of HSA-bound curcumin. At 0.3 curcumin/HSA molar ratio, typical molar CD values are Δε 496.6 nm +40 M −1 cm −1 and Δε 426.8 nm −40 M −1 cm −1 , respectively (pH 9.0, t=37 °C). The induced optical activity is attributed to a bent, right-handed chiral conformation of the HSA-bound curcumin molecule within which intramolecular exciton coupling occurs between the electric dipole transition moments of the dissymmetrically juxtaposed feruloyl chromophores. Deprotonation of phenolic OH group(s) of curcumin seems to be the reason leading to the conformational alteration of HSA-bound curcumin.

薬物とタンパク質との非共有結合・共有結合複合体のＥＳＩ／ＭＳ

ESI/MS of the noncovalently and covalently bound complexes of drugs with protein is useful to study on pharmacological effect or toxicological symptom of drugs because of solving the problem with far greater easier by MS than any other method. ESI/MS shows that protein folding of FKBP generates a native three-dimensional structure with multiple charged ions (+6~+8) at pH 6.8 from a denature linear one-dimensional structure with multiple charged ions (+10~+20) at pH 3.0. The noncovalently bound complex of FKBP-FK506 (12.6 kd) is observed a little in mixed solution of FK506 (0.8 kd) and FKBP (11.8 kd) at pH 3 or pH 6.8 by ESI/MS and its deconvoluted MS. But much amount of FKBP-FK506 (12.6 kd) generate during folding of FKBP after the pH of the solution containing FKBP and FK506 changes from pH 3.0 to pH 6.8. Drug A is a large ring compound with aminal bond and produced the covalent complex with human serum albumin (DA=HSA) that was measured easily by LC/ESI/MS and its deconvoluted mass. The stoichiometry of ratio (Drug : HSA) was shown by molecular ions at m/z 67,610 (1:1 DA=HSA complex), 68,792 (2:1 DA=HSA complex), 70,187 (3:1 DA=HSA complex). The main metabolites acyl glucronides of Drug B and C produced the covalent binding complex with HSA (DB=HSA and DC=HSA) that were easily measured by post-averaging scan of LC/ESI/MS and its deconvoluted MS. These data are useful to improve the post candidate and decide stop and continue of development.

Photophysics of Hypericin and Hypocrellin A in Complex with Subcellular Components: Interactions with Human Serum Albumin

Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and approximately 5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbonyl oxygen and the N1-H of the tryptophan residue in the IIA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of approximately 3 x 10(-15) s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the approximately 5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.

Amphotericin B as an intracellular antioxidant: Protection against 2,2′-azobis(2,4-dimethylvaleronitrile)-induced peroxidation of membrane phospholipids in rat aortic smooth muscle cells

The antifungal activity of amphotericin B (AmB) and its side-effects (e.g. nephrotoxicity and hemolytic action) are suggested to be associated with its prooxidant effects in target cells. To test this hypothesis, we have undertaken studies to examine the role of AmB in oxidative stress in cultured rat aortic smooth muscle cells (SMC) incubated in the absence or in the presence of a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). No changes in the pattern of membrane phospholipids could be detected by two-dimensional high performance thin-layer chromatography (HPTLC) after oxidative stress induced by AMVN in which the cells remained viable, as judged by trypan blue exclusion. To improve the sensitivity of detection of oxidative stress in the cells, cis-parinaric acid (PnA) was incorporated biosynthetically into the membrane phospholipids [using PnA-human serum albumin (hSA) complex]. Incubation of the cells under aerobic conditions in the presence of up to 10 μM AmB showed no significant change in the pattern of PnA-labeled phospholipids, suggesting that AmB was not affecting the oxidative state of the cells. In contrast, treatment with AMVN (0.5 mM, incubation in the dark for 2 hr at 37 ° — conditions in which the viability of the cells was maintained) caused a significant reduction of all fluorescently labeled phospholipid fractions separated by HPLC. When PnA-labeled cells were subjected to oxidative stress by incubation with 0.5 mM AMVN in the presence of AmB, the loss of fluorescent phospholipids was reduced in a concentration-dependent manner over a concentration range of 0.25 to 10 μM. Thus, AmB does not produce any prooxidant effect but rather acts as an intracellular antioxidant.

In-situ UV-visible spectroelectrochemical and circular dichroic electrochemical study of bilirubin and bilirubin + human serum albumin complex

The electro-oxidation of bilirubin (BR) in aqueous solution was investigated by cyclic voltammetry and in-situ thin-layer spectroelectrochemical techniques. It was found that both oxidation processes of BR are two electron transfer reactions. A mechanism for the electro-oxidation of BR in aqueous solution has been proposed. The electro-oxidation behavior of bilirubin + human serum albumin (BR + HSA) complex was also studied. Conformational changes in the BR + HSA complex during electro-oxidation, as reviewed by in-situ circular dichroism (CD) electrochemistry, are reported for the first time. A mechanism for the changes in the CD spectra of the complex during electro-oxidation is proposed.

Structural studies on metal-serum albumin I. an ultraviolet spectroscopic study of copper(II)-human serum albumin complexes

It has been reported for the first time that the concentration of human serum albumin (HAS) has a notable impact on the structure of Cu(II)-HSA complexes. Two configurations of Cu(II)-HSA have been found in the range of experimental concentrations used and the formation conditions and transformation processes of these two configurations have been studied by ultraviolet spectroscopy. The optical electronegativities of Cu(II) and relevant ligand atoms have also been calculated and discussed.

279 Nickel antibodies react with a Ni 2+/HSA complex

279 Nickel antibodies react with a Ni 2+/HSA complex

Electron paramagnetic resonance and optical spectroscopic study of the heme–human serum albumin complex

Heme–human serum albumin (heme–HSA) complex is paramagnetic because of the presence of a Fe 3+ ion chelated into the protoporphyrin core [1, 2]. At 77 K or lower, the ferric heme in the complex is characterized by a high spin (s = 5 2 electronic configuration. However, the tetragonal symmetry usually displayed by high spin heme iron which is responsible for two EPR lines situated at g⊥ ≅ 6 and g∥ ≅ 2 (fig. 1a) appears to be rhombically distorted upon binding by HSA (Fig. 1b). This distortion shows splitting of the g⊥ ≅ 6 resonance into two resolvable g-values, g x and g y (insert of Fig. 1). The EPR spectrum is interpreted in terms of the spin hamiltonian: H = DS 2 z − 1 3S(S + 1) + E(S 2 x − S 2 y) + β eH̄ ·ĝ· S̄ where E/D = (g x − g y)/48 is, in the present case, equal to 5.35 × 10 −3, corresponding to a rhombicity [3] of 1.61%. Departure from tetragonality may be brought about by several reasons, e.g. by mechanical distortion of heme, perturbation of the heme π-electron distribution, or by π-electron binding to the iron at the 5th and 6th ligand positions [3]. ▪ ▪ ▪ Very little is known about the molecular structure of the complex and, in particular, about the heme or HSA functional groups involved in the binding. However, engagement of the axial positions of heme iron in the binding has been questioned and the hypothesis that the propionyl side chains are involved in this binding has been put forward [4, 5]. Our study is devoted to gain further information on the molecular aspects of the interaction between heme and HSA, in order to understand why HSA binds so tightly the heme groups (causing distortion of the iron symmetry), although it accomplishes a carrier function. The following results were obtained: 1. Absorption optical spectroscopy, in the wavelength range between 300 and 1100 nm, of heme alone (Fig. 2b) and of heme–HSA complex (Fig. 2a) in DMSO/H 2O solvent (in this solvent it has been ▪shown that hemes are monomeric and HSA is not denaturated [4]) permitted us to ascertain definitively that at least one axial position of iron is involved in the binding; 2. Spin labeling of the lone sulfhydryl group at position 34 of the protein, by a maleimide nitroxide (Fig. 3), led us to estimate using Leigh's theory a a distance of about 10 Å between the paramagnetic ferric heme ion and the bound spin label; 3. The use of Fe 3+-protoporphyrin, spin labeled at its propionyl carboxyl groups, provided strong evidence that these groups are directly involved in the formation of the complex; probably via hydrogen bonds. 4. Emission optical spectroscopy showed that progressive quenching of the lone tryptophan (at position 214) fluorescence occurred during the formation of the complex (Fig. 4). According to the expression 6: ▪ the distance r between the heme group and tryptophan was estimated to be very close to 1.5 nm. Taking into account these results, and the fact that KCN converted the complex to a low spin form only at high concentrations, we can imagine that the heme group is bound in a rather hydrophobic pocket to HSA, which provides a ligand to the 5th axial position of iron and further hydrogen bonds to the propionyl side chains. The pocket is situated very close to the SH group and the close-lying aromatic tryptophan is probably responsible for the rhombic distortion, via a perturbation of the heme π-electron distribution [7]. Further thermodynamic results suggest the occurrence of an entropy-controlled internalization process of the bound heme, to provide steric protection against diffusion off the carrier protein surface, thus allowing for efficient release only at the target.