DNA interaction with human serum albumin studied by affinity capillary electrophoresis and FTIR spectroscopy

The question addressed in this study is how does the protein-DNA complexation affect the structure and dynamics of DNA and protein in aqueous solution. We examined the interaction of calf-thymus DNA with human serum albumin (HSA) in aqueous solution at physiological conditions, using constant DNA concentration of 12.5 mM (phosphate) and various HSA contents 0.25 to 2% or 0.04 to 0.3 mM. Affinity capillary electrophoresis and FTIR spectroscopic methods were used to determine the protein binding mode, the association constant, sequence preference, and the biopolymer secondary structural changes in the HSA-DNA complexes. Spectroscopic evidence showed two types of HSA-DNA complexes with strong binding of K(1) = 4.5 x 10(5) M(-1) and weak binding of K(2) = 6.10 x 10(4) M(-1). The two major binding sites were located on the G-C bases and the backbone PO(2) group. The protein-DNA interaction stabilizes the HSA secondary structure. A minor alteration of B-DNA structure was observed, while no major protein conformational changes occurred.     

Resveratrol binding to human serum albumin

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419-426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclooxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents (microM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure. Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K(Res) = 2.56 x 10(5) M(-1). The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of alpha-helix from 57% (free HSA) to 62% and a decrease of beta-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.     

Interaction of taxol with human serum albumin

Taxol (paclitaxel) is an anticancer drug, which interacts with microtuble proteins, in a manner that catalyzes their formation from tubulin and stabilizes the resulting structures (Nogales et al., Nature 375 (1995) 424-427). This study was designed to examine the interaction of taxol with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 0.0001-0.1 mM, and HSA (fatty acid free) concentration of 2% w/v. Gel electrophoresis, absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second-derivative resolution enhancement were used to determine the drug binding mode, binding constant and the protein secondary structure in the presence of taxol in aqueous solution. Spectroscopic evidence showed that taxol-protein interaction results into two types of drug-HSA complexes with overall binding constant of K=1.43 x 10(4) M(-1). The molar ratios of complexes were of taxol/HSA 30/1 (30 mM taxol) and 90/1 (90 mM taxol) with the complex ratios of 1.9 and 3.4 drug molecules per HSA molecule, respectively. The taxol binding results in major protein secondary structural changes from that of the alpha-helix 55 to 45% and beta-sheet 22 to 26%, beta-anti 12 to 15% and turn 11 to 16%, in the taxol-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of taxol in aqueous solution.     

Retinol and retinoic acid bind human serum albumin: stability and structural features

Vitamin A components, retinol and retinoic acid, are fat-soluble micronutrients and critical for many biological processes, including vision, reproduction, growth, and regulation of cell proliferation and differentiation. The cellular uptake of Vitamin A is through specific interaction of a plasma membrane receptor with serum retinol-binding protein. Human serum albumin (HSA), as a transport protein, is the major target of several micronutrients in vivo. The aim of present study was to examine the interaction of retinol and retinoic acid with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various retinoid contents. FTIR, UV–vis, CD and fluorescence spectroscopic methods were used to determine retinoid binding mode, the binding constant and the effects of complexation on protein secondary structure.

Structural analysis showed that retinol and retinoic acid bind non-specifically (H-bonding) via protein polar groups with binding constants of K_ret = 1.32 (±0.30) × 10⁵ M⁻¹ and K_retac = 3.33 (±0.35) × 10⁵ M⁻¹. The protein secondary structure showed no alterations at low retinoid concentrations (0.125 mM), whereas at high retinoid content (1 mM), an increase of -helix from 55% (free HSA) to 60% and a decrease of β-sheet from 22% (free HSA) to 18% occurred in the retinoid–HSA complexes. The results point to a partial stabilization of protein secondary structure at high retinoid content.     

Resveratrol Binding to Human Serum Albumin

Abstract

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419–426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclo- oxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents μM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure.

Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K_Res = 2.56× 10⁵ M^?1. The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of α-helix from 57% (free HSA) to 62% and a decrease of β-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.     

The effects of poly(ethylene glycol) on the solution structure of human serum albumin

Protein physical and chemical properties can be altered by polymer interaction. The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many organic and polymer molecules. This study was designed to examine the interaction of HSA with poly(ethylene glycol) (PEG) in aqueous solution at physiological conditions. Fourier transform infrared, ultraviolet-visible, and CD spectroscopic methods were used to determine the polymer binding mode, the binding constant, and the effects of polymer complexation on protein secondary structure.The spectroscopic results showed that PEG is located along the polypeptide chains through H-bonding interactions with an overall affinity constant of K = 4.12 x 10(5) M(-1). The protein secondary structure showed no alterations at low PEG concentration (0.1 mM), whereas at high polymer content (1 mM), a reduction of alpha-helix from 59 (free HSA) to 53% and an increase of beta-turn from 11 (free HSA) to 22% occurred in the PEG-HSA complexes (infrared data). The CDSSTR program (CD data) also showed no major alterations of the protein secondary structure at low PEG concentrations (0.1 and 0.5 mM), while at high polymer content (1 mM), a major reduction of alpha-helix from 69 (free HSA) to 58% and an increase of beta-turn from 7 (free HSA) to 18% was observed.     

Human serum albumin complexes with chlorophyll and chlorophyllin

Porphyrins and their metal derivatives are strong protein binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA and protein. The presence of several high-affinity binding sites on human serum albumin (HSA) makes it possible target for many organic and inorganic molecules. Chlorophyll a and chlorophyllin (a food-grade derivative of chlorophyll), the ubiquitous green plant pigment widely consumed by humans, are potent inhibitors of experimental carcinogenesis and interact with protein and DNA in many ways. This study was designed to examine the interaction of HSA with chlorophyll (Chl) and chlorophyllin (Chln) in aqueous solution at physiological conditions. Fourier transform infrared, UV-visible, and CD spectroscopic methods were used to determine the pigment binding mode, the binding constant, and the effects of porphyrin complexation on protein secondary structure. Spectroscopic results showed that chlorophyll and chlorophyllin are located along the polypeptide chains with no specific interaction. Stronger protein association was observed for Chl than for Chln, with overall binding constants of K(Chl) = 2.9 x 10(4)M(-1) and K(Chln) = 7.0 x 10(3)M(-1). The protein conformation was altered (infrared data) with reduction of alpha-helix from 55% (free HSA) to 41-40% and increase of beta-structure from 22% (free HSA) to 29-35% in the pigment-protein complexes. Using the CDSSTR program (CD data) also showed major reduction of alpha-helix from 66% (free HSA) to 58 and 55% upon complexation with Chl and Chln, respectively.     

Polyamine analogues bind human serum albumin

Polyamine analogues show antitumor activity in experimental models, and their ability to alter activity of cytotoxic chemotherapeutic agents in breast cancer is well documented. Association of polyamines with nucleic acids and protein is included in their mechanism of action. The aim of this study was to examine the interaction of human serum albumin (HSA) with several polyamine analogues, such as 1,11-diamino-4,8-diazaundecane (333), 3,7,11,15-tetrazaheptadecane.4HCl (BE-333), and 3,7,11,15,19-pentazahenicosane.5HCl (BE-3333), in aqueous solution at physiological conditions using a constant protein concentration and various polyamine contents (microM to mM). FTIR, UV-visible, and CD spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind nonspecifically (H-bonding) via polypeptide polar groups with binding constants of K333 = 9.30 x 10(3) M(-1), KBE-333 = 5.63 x 10(2) M(-1), and KBE-3333 = 3.66 x 10(2) M(-1). The protein secondary structure showed major alterations with a reduction of alpha-helix from 55% (free protein) to 43-50% and an increase of beta-sheet from 17% (free protein) to 29-36% in the 333, BE-333, and BE-3333 complexes, indicating partial protein unfolding upon polyamine interaction. HSA structure was less perturbed by polyamine analogues compared to those of the biogenic polyamines.     

Analysis of binding interaction between the natural apocarotenoid bixin and human serum albumin by circular dichroism and fluorescence spectroscopy

Bixin is an important, pharmacologically active dietary cis-carotenoid, but its interaction with potential macromolecular targets is completely unexplored. This work was aimed to study the binding of bixin to human serum albumin (HSA), the most abundant protein in blood plasma. Circular dichroism (CD) spectroscopy in combination with UV/VIS absorption spectroscopy and fluorescence quenching techniques were applied. Appearance of induced CD bands in the UV- and VIS-absorption spectral regions indicated the formation of non-covalent carotenoid-albumin complexes. Shape and spectral position of the extrinsic Cotton effects suggested the binding of a single bixin molecule to HSA in chiral conformation. Scatchard and non-linear regression analyses of CD titration data resulted in similar values for the association constant (Ka = 6.6 and 4.6x10(5) M(-1), resp.) and for the number of binding sites (n = 1). The binding interaction was independently confirmed by fluorescence-quenching experiment from which the binding parameters were also calculated. CD Displacement measurements performed with marker ligands established that the main drug binding sites of HSA are not involved in binding of bixin. Palmitic acid decreased the amplitude of the induced CD bands suggesting a common albumin binding site for bixin and long-chain fatty acids. The above data indicate that HSA plays a significant role in the plasma transportation of bixin and related dietary carboxylic acid carotenoids.     

Influence of aspirin and iron(III) tetrasulfonated phthalocyanine on bilirubin binding by human serum albumin

The interaction of bilirubin with aspirin-modified human serum albumin (HSA) and the influence of iron tetrasulfonated phthalocyanine on bilirubin binding by the native protein has been studied by difference spectroscopy and circular dichroism measurements. Spectroscopic studies of the systems containing bilirubin and aspirin-modified HSA compared to the analogous systems with the native protein have shown that selective acetylation of albumin at lysine 199 inhibits bilirubin binding by this protein. In both cases, interaction between bilirubin and albumin leads to complex formation at a molar ratio of ligand to protein of 2:1. The studies of the reaction of bilirubin with fragments of albumin produced by reaction with CNBr have demonstrated that one of the strong bilirubin binding sites is located in the M fragment and is close to the high-affinity binding site of aspirin. The other one was found in fragment C. Acetylation of albumin brings about marked conformational change in the protein, which probably accounts for the decrease in its ability to react with anti-HSA antibody. Bilirubin does not change the secondary structure of albumin but, like aspirin, lowers its antigenicity. It has been suggested that the decrease in antigenic properties in this case results from cooperation of the closely neighboring antigenic and bilirubin-binding sites. The studies of the influence of iron(III) tetrasulfonated phthalocyanine on bilirubin binding by HSA suggest that there is no competition between strong sites for iron(III) tetrasulfonated phthalocyanine and bilirubin, but these compounds compete for some of the weaker sites.     

Human serum albumin interaction with formononetin studied using fluorescence anisotropy, FT-IR spectroscopy, and molecular modeling methods

Interaction of formononetin with a model transport protein, human serum albumin (HSA), has been studied using fluorescence anisotropy, FT-IR spectroscopy, and molecular modeling methods. Upon binding with HSA, the fluorescence spectrum of formononetin exhibits appreciable hypsochromic shift along with an enhancement in the fluorescence intensity. Gradual addition of HSA led to a marked increase in fluorescence anisotropy (r). From the value of fluorescence anisotropy, it is argued that the drug is located in a restricted environment of protein. The binding constant (K approximately 1.6 x 10(5) M(-1)) and the standard free energy change (DeltaG(0) approximately -29.9 kJ/mol) of formononetin-HSA interaction have been calculated according to the relevant fluorescence data. Fourier transform infrared measurements have shown that the secondary structures of the protein have been changed by the interaction of formononetin with HSA. Computational mapping of the possible binding sites of formononetin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA.     

人血清白蛋白与镍离子相互作用的热力学研究

1　Introduction　　Serumalbuminproteinsareamongthemosthighlystudiedandappliedinbiochemistry[1～ 4].Albuministhemostabundantproteininbloodplasmaandoneofitsmainfunctionsisbasedonauniqueabilitytobindnumerousendogenousandexogenouscompounds.Duetoitsligandbindingpropertiesalbuminservesasacirculatingdepotofsomemetabolites.Thisdepoteffectisoftenmadeuseofindrugtherapy.　　Humanserumalbumin(HSA)isasinglepeptidechainconsistingof 5 85aminoacids( 6 6 5ku)asdeterminedbyaminoacidsequencestudies[5] andasde…     

Interactions of atrazine and 2,4-D with human serum albumin studied by gel and capillary electrophoresis, and FTIR spectroscopy.

The herbicides 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine (atrazine) and 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used in agricultural practice to fight dicotyledon weeds mainly in maize, cereals, and lucerne. As a result, these compounds are found not only in the plants, soil, and water, but also in the cultivated ground in the following years as well as in agricultural products such as fruits, milk, butter, and sugar beet. The toxicological effects of herbicides occur in vivo, when transported to the target organ through the bloodstream. It has been suggested that human serum albumin (HSA) serves as a carrier protein to transport 2,4-D to molecular targets. This study was designed to examine the interaction of atrazine and 2,4-D with HSA in aqueous solution at physiological pH with herbicide concentrations of 0.0001-1 mM, and final protein concentration of 1% w/v. Gel and capillary electrophoresis, UV-visible and Fourier transform infrared spectroscopic methods were used to determine the drug binding mode, the drug binding constant, and the protein secondary structure in aqueous solution. Structural analysis showed that different types of herbicide-HSA complexes are formed with stoichiometric ratios (drug/protein) of 3:1 and 11:1 for atrazine and 4.5:1 and 10:1 for 2,4-D complexes. Atrazine showed a weak binding affinity (K=3.50 x 10(4) M(-1)), whereas two bindings (K(1)=2.50 x 10(4) M(-1) and K(2)=8.0 x 10(3) M(-1)) were observed for 2,4-D complexes. The herbicide binding results in major protein secondary structural changes from that of the alpha-helix 55% to 45--39% and beta-sheet 22% to 24--32%, beta-anti 12% to 10--22% and turn 11% to 12--15%, in the drug-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of herbicides in aqueous solution.     

Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments

Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.     

Interaction of anesthetic supplement thiopental with human serum albumin

Thiopental (TPL) is a commonly used barbiturate anesthetic. Its binding with human serum albumin (HSA) was studied to explore the anesthetic-induced protein dysfunction. The basic binding interaction was studied by UV-absorption and fluorescence spectroscopy. An increase in the binding affinity (K) and in the number of binding sites (n) with the increasing albumin concentration was observed. The interaction was conformation-dependent and the highest for the F isomer of HSA, which implicates its slow elimination. The mode of binding was characterized using various thermodynamic parameters. Domain II of HSA was found to possess a high affinity binding site for TPL. The effect of micro-metal ions on the binding affinity was also investigated. The molecular distance, r, between donor (HSA) and acceptor (TPL) was estimated by fluorescence resonance energy transfer (FRET). Correlation between the stability of the TPL-N and TPL-F complexes and drug distribution is discussed. The structural changes in the protein investigated by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy reflect perturbation of the albumin molecule and provide an explanation for the heterogeneity of action of this anesthetic.     

Ultraviolet-circular dichroism spectroscopy and potentiometric study of the interaction between human serum albumin and sodium perfluorooctanoate

The interaction of a fluorinated surfactant, sodium perfluorooctanoate, with human serum albumin (HSA) has been investigated by a combination of ultraviolet-circular dichroism (UV-CD) spectroscopy and potentiometry (by a home-built ion-selective electrode) techniques to detect and characterize the conformational transitions of HSA. By using difference spectroscopy, the transition was followed as a function of temperature, and the data were analyzed to obtain the parameters characterizing the thermodynamics of unfolding. The results indicate that the presence of surfactant drastically changes the melting unfolding, acting as a structure stabilizer and delaying the unfolding process. Potentiometric measurements were used to determine the binding isotherms and binding capacity for this system. The isotherm shows a high affinity of surfactant molecules for HSA. The average number of surfactant molecules absorbed per protein molecule (at 28 mM of surfactant concentration) was found to be approximately 900, about 6 g of surfactant per gram of protein. The shape of the binding capacity curve and the relation between binding capacity and extend of cooperativity were examined. From these analysis, the values of g (number of ligand-binding sites), KH (Hill binding constant), and nH (Hill coefficient) were determined.     

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 K(Pb-HSA)?=?8.2 (±0.8)×10(4) M(-1) and K(Pb-BSA)?=?7.5 (±0.7)×10(4) 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.     

Binding of 3-carbethoxipsoralen to human serum albumin and human serum: influence of free fatty acids

Characteristics of the binding of 3-carbethoxipsoralen (3CPS) to human serum albumin (HSA) and serum proteins have been studied. An electrophoretic study showed that the predominant binding protein fraction was albumin, with small binding to globulins. Binding to HSA, studied by equilibrium dialysis, is 75% and characterized by a small saturable number of binding sites (N = 0.27) with a moderate affinity constant (K = 8 X 10(4) M-1). Free fatty acids were shown to decrease 3CPS binding to HSA by a non competitive process.