Crystal and molecular conformation of the fluorescent probe. 8-anilino-1-naphthalenesulfonic acid (ANS).

The crystal and molecular structure of the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS) has been determined as the ammonium monohydrate with two conformationally distinct molecules in the triclinic $P\overline{1}$ lattice. The angles between the aromatic rings and the CNC plane are ?9/22° and 112/22° respectively. There is an NH...O intramolecular hydrogen bond in each molecule indicating that hydrogen bond formation is not dependent on the anilino geometry. There are also short intramolecular H...H contacts involving the hydrogens which have anomalous proton shifts shown in a recent NMR study.     

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

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

Reversible binding of ethacrynic acid to human serum albumin: Difference circular dichroism study

The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anticooperative) and to site III (cooperative). Chirality 11:33–38, 1999. © 1999 Wiley‐Liss, Inc.     

Interaction of diiodo-L-tyrosine and triiodophenol with bovine serum albumin. Circular dichroism and fluorescence studies.

As a model study to investigate the binding mechanism between thyroid hormones and carrier protein, the interaction of diiodo-L-tyrosine (DIT) and triiodophenol (I3phi) with bovine serum albumin (BSA) was investigated by circular dichroism (CD) and fluorescence methods. In both the DIT-BSA system and the I3phi-BSA system, induced Cotton effect was observed in the wavelength region near 320 nm. This induced Cotton effect was measured at various molar ratios of ligands to BSA (L/P). The value of the ellipticity at 319 nm, [theta]319, in the I3phi-BSA system was remarkably large compared with that of the DIT-BSA system, and [theta]319 at an L/P ratio of one was -1.96 X 10(4) (degree cm2 decimole-1) for the I3phi-BSA system and -0.1 X 10(4) for the DIT-BSA system. The binding constants for the combination of BSA with a single molecule of ligand, calculated by measuring the quenching of the fluorescence of the protein, were 1.33 X 10(5) M(-1) at 15 degrees for the DIT-BSA system and 1.6 X 10(9) M(-1) at 28 degrees for the I3theta-BSA system. These results suggest that the binding of I3theta to BSA is stronger than that of DIT and a cleft may exist more congruent with the molecular dimensions of I3theta than with those of DIT.     

Surface characterization of human serum albumin and sodium perfluorooctanoate mixed solutions by pendant drop tensiometry and circular dichroism

The interfacial behavior of mixed human serum albumin (HSA)/sodium perfluorooctanoate (C8FONa) solutions is examined by using two experimental techniques, pendant drop tensiometry and circular dichroism spectroscopy. Through the analysis of the surface tension of the mixed solutions, surface competitive adsorption at the air-water interface between C8FONa and HSA is detected. The dynamic adsorption curves exhibit the distinct regimes in their time-dependent surface tension. The nature of these regimes is further analyzed in terms of the variation of the molecules surface areas. As a consequence, a compact and dense structure was formed where protein molecules were interconnected and overlapped. Thus, a reduction of the area occupied per molecule from 100 to 0.2 nm(2) is interpreted as a gel-like structure at the surface. The presence of the surfactant seems to favor the formation of this interfacial structure. Finally, measurements of circular dichroism suggests a compaction of the protein due to the association with the surfactant given by an increase of alpha-helix structure in the complexes as compared to that of pure protein.     

A circular dichroism and fluorescence quenching study of the interactions between rhodium(II) complexes and human serum albumin.

Various divalent rhodium complexes Rh2(L)4 (L = acetate, propionate, butyrate, trifluoroacetate and trifluoroacetamidate) have been found to bind to non-defatted human serum albumin (HSA) at molar ratios about 8:1. The circular dichroism measurements showed that the more liposoluble carboxylates, butyrate and trifluoroacetate, caused the major alterations of the secondary structure of HSA. Stern-Volmer constants for the fluorescence quenching of the buried Trp214 residue by these complexes were also higher for the lipophilic metal compounds. In the case of the rhodium carboxylates it was observed that their denaturating and quenching properties could be explained in terms of their liposolubilities: the higher their lipophilic characters, the higher their abilities to penetrate inside the protein framework leading to structural alterations, and the closer they could get to the Trp residue causing fluorescence quenching. The liposoluble amidate complex, Rh2 (tfc)4, presented an intermediate quenching and did not cause structural alterations in the protein, presumably not penetrating inside the peptidic backbone. This study shows that it is possible to design new antitumor metal complexes which bind, to a large extent, to a transport protein causing little structural damage.     

The binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin.

Binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin are reported. The critical micelle concentration (CMC) and the limiting solubility of 5-doxyl stearic acid were determined using the electron spin resonance (ESR)-spin label method. The CMC and the limiting solubility of this spin-label stearic acid in saline-phosphate buffer are 3.5 x 10(-5) M and 2 x 10(-4) M, respectively. We found no ESR line width evidence for pre-association of the spin-label stearate below the CMC. Maximum binding of the spin-label stearate to both bovine and human albumin occurs before micelle formation. The binding isotherm for spin-label stearic acid interaction with bovine albumin is in agreement with data obtained by others using [1-(14)C]stearic acid. For human albumin, comparison is difficult since previous data obtained with [1-(14)C]stearic acid vary widely. Comparison of the ESR 2T(||) values (the splitting between low and high field extremes, a measure of the degree of immobilization of protein-bound spin-label stearate) for bovine and human albumin indicates a greater immobilization of the spin-label molecules bound to human albumin. The binding data indicate that complexes are formed with bound spin-label stearate/albumin ratios of at least 18. The computed equilibrium constants for both bovine and human albumin indicate that the first seven spin-label molecules are tightly bound, log K > 5.0. The species predicted to form in solution by these equilibrium constants are reported.     

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.     

Investigation on the effect of fluorescence quenching of bovine serum albumin by cefoxitin sodium using fluorescence spectroscopy and synchronous fluorescence spectroscopy

The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of the synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanisms of drugs with protein and was consistent with one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between drug and protein and energy‐transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role in the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. The primary binding site for cefoxitin sodium was sub‐hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill's coefficients (n_H) was approximately equal to 1, which suggested no cooperativity in the bovine serum albumin–cefoxitin sodium system. The donor‐to‐acceptor distance r < 7 nm indicated that static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non‐radiation energy‐transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2015 John Wiley & Sons, Ltd.     

Kinetics of stearic acid transfer between human serum albumin and sterically stabilized liposomes

The kinetics of the transfer of stearic acids between human serum albumin (HSA) and long circulating sterically stabilised liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of the polymer-lipid poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE) have been studied by fluorescence spectroscopy. The study exploits the fact that HSA has a single tryptophan (Trp) residue and that the intrinsic Trp-emission intensity is quenched by the presence of doxyl spin-labelled stearic acids (SASL). Protein/lipid dispersions are considered in which SASL molecules are inserted either in the protein or in the SSL, and the transfer of SASL between the protein and SSL is conveniently monitored by the time variation of the inherent Trp-fluorescence intensity of HSA. It was found that the transfer of fatty acids between HSA and SSL depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers) and on the grafting density of the PEG-lipids at the lipid/protein interface. In the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer, and it is higher for the passage from DPPC bilayers to HSA. The presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer chains, especially at low grafting density and for lipid membranes in the fluid phase.     

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.     

The electrochemically induced conformational transition of disulfides in bovine serum albumin studied by thin layer circular dichroism spectroelectrochemistry

The conformational transition of disulfides in bovine serum albumin (BSA) induced by electrochemical redox reaction of disulfides were monitored by in-situ circular dichroism (CD) spectroelectrochemistry, with a long optical path thin layer cell and analyzed by a singular value decomposition least square (SVDLS) method. Electrochemical reduction of disulfides drives the left-handed conformation of disulfides changed into the right-handed. At open circuit, eight of the 17 disulfides were of left-handed conformation. Four of the 17 disulfides took part in the electrochemical reduction with an EC mechanism. Only one-fourth of the reduced disulfides returned to left-handed conformation in the re-oxidation process. Some parameters of the electrochemical reduction process, i.e. the number of electrons transferred and electron transfer coefficient, n = 8, alpha n = 0.15, apparent formal potential, E1(0') = -0.65(+/-0.01) V, standard heterogeneous electron transfer rate constant, k1(0) = (2.84 +/- 0.14) x 10(-5) cm s(-1) and chemical reaction equilibrium constant, Kc = (5.13 +/- 0.12) x 10(-2), were also obtained by double logarithmic analysis based on the near-UV absorption spectra with applied potentials.     

Understanding bilirubin conformation and binding. Circular dichroism of human serum albumin complexes with bilirubin and its esters

Human serum albumin binds tightly and noncovalently to a wide variety of hydrophobic bilirubins, including (4Z,15Z)-bilirubin-IX alpha, its dimethyl ester and mono methyl esters, its mono 2-butyl esters and amides, the dimethyl ester of (4Z,15Z)-mesobilirubin-IV alpha, and even (4Z,15Z)-etiobilirubin-IV gamma. The heteroassociation complexes formed from these highly water-insoluble pigments and the protein can be prepared in pH 7.4 aqueous by using a small quantity of dimethyl sulfoxide as amphiphilic carrier. In those solutions the protein acts as a water-soluble chiral complexation agent to induce an asymmetric transformation of the bound pigment. This is recognized by positive chirality, bisignate induced circular dichroism (CD) Cotton effects that fall in the region of the bichromophoric pigment's long wavelength UV-visible absorption band and are characteristic of intramolecular exciton coupling of the bilirubin component pyrromethenone chromophores. The same-signed CD spectra shared by all the pigments of this work indicate selection at the protein binding site for a positive chirality conformer and suggest a common binding site. The CD intensities, which are greatest ([delta epsilon[ congruent to 50) for pigments with one or two free carboxyl groups, are consistent with a binding model where one salt linkage plays a major role in the enantioselectivity of the right-handed folded conformation stabilized by inter- and intramolecular hydrogen bonds.     

The effect of guanidine hydrochloride on the conformation of bovine pancreatic DNAase I as measured with circular dichroism

The denaturation of bovine pancreatic DNAase I (EC 3.1.21.1) by guanidine hydrochloride (GdnHCl) has been investigated with circular dichroism in the presence and absence of 1 mM Ca2+ at the wavelength region of 210-240 nm at 12.25 and 36 degree C. The change of the molar ellipticity at 220 nm by GdnHCl titration showed cooperative transition at each temperature and the midpoints of the titrations occurred near 2 M GdnHCl. At each temperature, the denaturation of DNAase I in the presence of 1 mM Ca2+ occurred a little slowly as compared with that in the absence of Ca2+. This suggests that 1 mM Ca2+ can to some extent stabilize the secondary structure of DNAase I against GdnHCl denaturation. The apparent free energy for the denaturation of DNAase I obtained by GdnHCl titration was calculated as 9.3 +/- 0.3 kcal/mol and 8.9 +/- 0.2 kcal/mol at 25 degree C in the presence and absence of 1 mM Ca2+, respectively. The possible regions for the alpha -helix and beta -structure of DNAase I were predicted from the amino acid sequence by probability calculation of Chou, P.Y. and Fasman, G.D., Adv. Enzymol. 47, 45-148. The characteristic feature is that the NH2-terminal half of DNAase I is rich in beta -structure and the COOH-terminal half contains mainly alpha -helix.