Effect of physiological concentration of urea on the conformation of human serum albumin

We report that the presence of very low concentrations (<0.1 M) of urea, a widely used chemical denaturant, induces structure formation in the water-soluble globular protein human serum albumin (HSA) at pH 7. We have presented results suggesting an almost 8% and 5% increase in alpha-helix in the presence of 10 mM urea (U) and 20 mM monomethylurea (MMU), respectively. Far and near-UV circular dichroism studies along with tryptophan fluorescence and 1-anilino-8-naphthalenesulphonicacid (ANS) binding support our view. We hypothesize that both U and MMU, at such low concentrations, modify the solvent structure, increase the dielectric constant and consequently increase hydrophobic forces resulting in enhanced alpha-helical content. The implications of these results of the lower urea regime are significant because the physiological blood urea ranges from 2.5 to 7.5 mM.     

Spectroscopic investigation on the interaction of J-aggregate with human serum albumin

The interactions of three cyanine dyes, which exhibit different meso substituent in polymethine chain, with human serum albumin (HSA) have been investigated by the means of absorption, fluorescence and circular dichroism (CD) spectra. In phosphate buffer solution (PBS), the mentioned dyes exist not as isolated monomers but rather in the formation of J-aggregation. In the presence of HSA, the absorption and fluorescence emission spectra indicated that the J-aggregation was decomposed to monomer because of the strong affinity between dye molecules and HSA. Besides the association of cyanine dyes with HSA, binding to HSA gave rise to the J-aggregation CD signals. The meso substituent in the polymethine plays an important role in the interaction of HSA and the J-aggregation. Spectral studies showed that the dye bound with HSA in a 1:1 formation. The apparent constant (K(a)) value was roughly identified by analysis of the corresponding fluorescence data at various HSA concentrations. The higher affinity of the molecule with meso phenyl towards HSA with respect to molecules with meso ethyl or methyl can be attributed to the arrangement of molecules in J-aggregation and the hydrophobic force between the molecules and HSA.     

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.     

The effects of drug complexation on the stability and conformation of human serum albumin

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.     

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.     

The subsequent effect of interaction between Co(2+) and human serum albumin or bovine serum albumin.

A notable hysteretic effect has been observed in the interaction of Co(II) with human serum albumin (HSA) or bovine serum albumin (BSA) using UV-Visible spectrometry at physiological pH (7.43), which shows that the binding between Co(II) and HSA or BSA may induce a slow transition of HSA or BSA from the conformation of weaker affinity for Co(II) to one of stronger affinity (A-B transition). The rate constants and activation parameters of this transition were measured and are discussed. It is inferred that such a conformation transition may occur due to the binding of the first Co(II) ion with the peptide segment of N-terminal residues 1-3, which results in a 'hinged movement' of the relatively hydrophobic 'valley' in the IA subdomain. This process leads to a slow conformational transition in the albumins, makes the other binding sites of Co(II) exposed, and shows a positive cooperativity effect. The LMCT (ligand-to-metal charge transition) bands of the Co(II)-HSA and Co(II)-BSA systems also show a kind of hypochromic effect featuring a dipole-dipole interaction mechanism. This phenomenon is rarely reported.     

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.     

Anion-binding centers of human serum albumin during an N-F conformation transition and in isolated albumin and blood serum

Fluorescent probe N-phenyl-1-amino-8-sulfonaphthalene (ANS) was used for studying pH-dependent structural N-F-transition in human serum albumin of two kinds: in commercial albumin and in natural blood serum. The kinetics of ANS fluorescence decay in albumin solutions was measured. There were found two types of the sites occupied by ANS in albumin under physiological conditions (pH 7.4). In the first binding site ANS fluorescence decay time was 16.6 +/- 0.3 nsec and it was not significantly changed at N-F transition (pH 4.0). In the second binding site the decay time was dependent on pH in commercial albumin and was not significantly changed in serum. In the second binding site there were individual differences of ANS decay time (4.3 +/- 0.6 nsec). The observed ANS fluorescence intensity enhancing (about 40-50%) in N-F transition may be explained by an increase of albumin binding sites capacity for ANS.     

Effects of podophyllin and 8-hydroxyquinoline on meiosis

The induced meiotic abnormalities as a result of sprayingVicia faba plants with aqueous saturated solutions of podophyllin and 8-hydroxyquinoline were studied. The 2 drugs induced the same types of abnormalities including lagging chromosomes, stickiness, and bridges. The main difference between the 2 agents was the induction of polyploid P.M.Cs. by 8-hydroxyquinoline.     

Cytotoxic palladium(II) complexes of 8-aminoquinoline derivatives and the interaction with human serum albumin

Palladium(II) complexes are potential antitumor metallodrugs for their chemical resemblance to platinum(II) complexes. Two palladium(II) complexes (1 and 2) in the formula of [PdLⁿCl] [L¹ = N-(tert-butoxycarbonyl)-l-methionine-N′-8-quinolylamide, L² = L-alanine-N′-8-quinolylamide] have been synthesized accordingly. The structures of the complexes were fully characterized by X-ray crystallography. The palladium(II) center in 1 is coordinated by two N atoms and an S atom from L¹ with one chloride anion as the leaving group; while that in 2 is coordinated by three N atoms from L² with one chloride anion as the leaving group. The interaction between complex 1 and human serum albumin (HSA) has been investigated using fluorescence and circular dichroism spectroscopies. The complex seems to react with HSA chiefly through hydrophobic and electrostatic interactions, and it does not alter the α-helical nature of HSA. The cytotoxicity of these complexes has been tested against the human cervical cancer (HeLa), human mammary cancer (MCF-7), and human lung cancer (A-549) cell lines. Complex 1 displays a cytotoxic activity comparable to that of cisplatin, but complex 2 is less active than cisplatin.     

A spectroscopic study of the interaction of isoflavones with human serum albumin

Genistein and daidzein, the major isoflavones present in soybeans, possess a wide spectrum of physiological and pharmacological functions. The binding of genistein to human serum albumin (HSA) has been investigated by equilibrium dialysis, fluorescence measurements, CD and molecular visualization. One mole of genistein is bound per mole of HSA with a binding constant of 1.5 +/- 0.2 x 10(5) m(-1). Binding of genistein to HSA precludes the attachment of daidzein. The ability of HSA to bind genistein is found to be lost when the tryptophan residue of albumin is modified with N-bromosuccinimide. At 27 degrees C (pH 7.4), van't Hoff's enthalpy, entropy and free energy changes that accompany the binding are found to be -13.16 kcal x mol(-1), -21 cal x mol(-1) K(-1) and -6.86 kcal x mol(-1), respectively. Temperature and ionic strength dependence and competitive binding measurements of genistein with HSA in the presence of fatty acids and 8-anilino-1-naphthalene sulfonic acid have suggested the involvement of both hydrophobic and ionic interactions in the genistein-HSA binding. Binding measurements of genistein with BSA and HSA, and those in the presence of warfarin and 2,3,5-tri-iodobenzoic acid and F?rster energy transfer measurements have been used for deducing the binding pocket on HSA. Fluorescence anisotropy measurements of daidzein bound and then displaced with warfarin, 2,3,5-tri-iodobenzoic acid or diazepam confirm the binding of daidzein and genistein to subdomain IIA of HSA. The ability of HSA to form ternery complexes with other neutral molecules such as warfarin, which also binds within the subdomain IIA pocket, increases our understanding of the binding dynamics of exogenous drugs to HSA.     

Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site

Human serum albumin (HSA) is known to exist as N (pH approximately 7), B (pH approximately 9), and F (pH approximately 3.5) isomeric forms and an equilibrium intermediate state (I) accumulate in the urea induced unfolding pathway of HSA around 4.8-5.2 M urea concentrations. These states displayed characteristic structure and functions. To elucidate the ciprofloxacin (CFX) binding behavior of HSA, the binding of ciprofloxacin with these conformational states of human serum albumin (HSA) has been investigated by fluorescence spectroscopy. The binding constant (K) for N, B, F, and I conformation of HSA were 6.92 x 10(5), 3.87 x 10(5), 4.06 x 10(5), and 2.7 x 10(5) M(-1) and the number of binding sites (n) were 1.26,1.21, 1.16, and 1.19, respectively. The standard free energy changes (DeltaGbinding(0)) of interaction were found to be -33.3 (N isomer), -31.8 (B isomer), -32 (F isomer), and -30.0 kJ mol(-1) respectively. By using unfolding pathway of HSA, domain II of HSA has been assigned to possess binding site of ciprofloxacin. Plausible correlation between stability of CFX-N and CFX-B complexes and drug distribution have been discussed. At plasma concentration of HSA fraction of free CFX, which contributes potential to its rate of transport across cell membrane, was found to be approximately 80% more for B isomers compared to N isomers of HSA. The conformational changes in two physiologically important isomers of HSA (N and B isomers) upon ciprofloxacin binding were evaluated by measuring far, near-UV CD, and fluorescence properties of the CFX-HSA complex.     

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.     

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.     

The role of configuration and conformation in the binding of 2,3-benzodiazepines to human serum albumin

2,3-Benzodiazepines containing a centre of asymmetry at C-5 possess both central and helical chiralities, and the solution of their racemates contains four molecular species. The binding of these compounds to human serum albumin (HSA) was studied by affinity chromatography. The binding strength depended both on the steric orientation of the 5-ethyl substituent and on the conformation of the diazepine ring. Conformation P (defined by the positive sign of C-1-N-2-N-3-C-4 torsion angle) is favoured, while the quasiaxial orientation of the 5-ethyl substituent is not favoured by the albumin molecule.     

On the hydrodynamics and temperature dependence of the solution conformation of human serum albumin from viscometry approach

The paper presents the results of viscosity determinations on aqueous solutions of human serum albumin (HSA) at a wide range of concentrations and at temperatures ranging from 5 to 45 degrees C. On the basis of a modified Arrhenius formula and Mooney's equation, the viscosity-temperature and viscosity-concentration dependence of the solutions are discussed. The effective specific volume, the activation energy and entropy of viscous flow for hydrated HSA were calculated. Different models of HSA molecule are discussed and the best one-from the hydrodynamic point of view-was established. At low concentration limit, such rheological quantities as the intrinsic viscosity and Huggins coefficient were obtained. Using the dimensionless parameter [eta]c, the existence of three characteristic ranges of concentrations: diluted, semi-diluted and concentrated, was shown.     

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine and human serum albumin

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine (CECT) and human serum albumin (HSA) were investigated using fluorescence, UV absorption spectroscopic and molecular modeling techniques under simulative physiological conditions. The intrinsic fluorescence intensity of HSA was decreased with the addition of CECT. The fluorescence data handled by Stern–Volmer equation proved that the quenching mechanism of the interaction between CECT and HSA was a static quenching procedure. The binding constants evaluated utilizing the Lineweaver–Burk equation at 17, 27 and 37?°C, were 2.340?×?10⁴, 2.093?×?10⁴ and 1.899?×?10⁴?L?mol^?1, respectively. The thermodynamic parameters were calculated according to van’t Hoff equations. Negative enthalpy (ΔH) and positive entropy (ΔS) values indicated that both hydrogen bond and hydrophobic force played a major role in the binding process of CECT to HSA, which was consistent with the results of the molecular modeling study. In addition, the effect of other ions on the binding constant of CECT-HSA was examined.