Evaluation of enantioselective binding of antihistamines to human serum albumin by ACE

The drug binding to plasma and tissue proteins is a fundamental factor in determining the overall pharmacological activity of a drug. HSA, together with alpha(1)-acid glycoprotein, are the most important plasma proteins, which act as drug carriers, with implications on the pharmacokinetic of drugs. Among plasma proteins, HSA possesses the highest enantioselectivity. In this paper, a new methodology for the study of enantiodifferentiation of chiral drugs with HSA is developed and applied to evaluate the possible enantioselective binding of four antihistamines: brompheniramine, chlorpheniramine, hydroxyzine and orphenadrine to HSA. This study includes the determination of affinity constants of drug enantiomers to HSA and the evaluation of the binding sites of antihistamines on the HSA molecule. The developed methodology includes the ultrafiltration of samples containing HSA and racemic antihistaminic drugs and the analysis of the free or bound drug fraction using the affinity EKC-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of antihistamines to HSA, the major plasmatic protein.     

FLUORESCENCE INVESTIGATIONS OF ALBUMIN FROM PATIENTS WITH FAMILIAL DYSALBUMINEMIC HYPERTHYROXINEMIA*

Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant syndrome in which clinically euthyroid patients have elevated total thyroxine levels. These high serum thyroxine levels are traceable to altered binding of thyroxine to the patient's albumin. Albumin from FDH patients and normal volunteers have been purified. Reverse-phase and ion-exchange high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the FDH-human serum albumin (HSA) samples show a single band that comigrates with normal HSA. In both protein solutions the intrinsic fluorescence, upon 280 nm excitation, is predominantly due to the single tryptophan residue. The quantum yield of this intrinsic fluorescence in the FDH-HSA solutions is, however, reduced relative to that of HSA. Furthermore, the “average” lifetime value of the tryptophan emission in the FDH-HSA sample is less than that of normal HSA, consistent with its reduced quantum yield. The binding of thyroxine to both albumins effectively quenches the tryptophan emission probably via a nonradiative energy transfer mechanism. Time-resolved data suggest that the albumin from the dysalbuminemic patients is actually an approximately equimolar mixture of normal HSA and FDH-HSA indicative of heterologous expression. Quenching of the intrinsic HSA and FDH-HSA fluorescence by serial additions of thyroxine showed enhanced quenching of FDH-HSA relative to HSA at any T4 to albumin mole ratio, therefore supporting earlier reports of increased thyroxine affinity to FDH-HSA.     

A Molecular Magnet Confined in the Nanocage of a Globular Protein

The effect of confinement and energy transfer on the dynamics of a molecular magnet, known as a model system to study quantum coherence, is investigated. For this purpose the well‐known polyoxovanadate [V₁₅As₆O₄₂(H₂O)]^6? (V₁₅) is incorporated into a protein (human serum albumin, HSA) cavity. Due to a huge overlap of the optical absorption spectrum of V₁₅ with the emission spectrum of a fluorescence center of HSA (containing a single tryptophan residue), energy transfer is induced and probed by steady‐state and time‐resolved fluorescence. The geometrical coordination and the distance of the confined V₁₅ to the tryptophan moiety of HSA are investigated at various temperatures. This effect is used as a local probe for the thermal denaturation of the protein at elevated temperatures.     

Betulinic acid binding to human serum albumin: a study of protein conformation and binding affinity

Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be K(BA)=1.685+/-0.01 x 10(6) M(-1), indicating a strong binding affinity. The secondary structure of the HSA-BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA+drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies.     

SPECTRAL STUDY OF THE PHOTOCHEMISTRY OF DIPYRROLE MODELS FOR BILIRUBIN BOUND TO HUMAN SERUM ALBUMIN

—Dipyrromethenones, which include xanthobilirubinic acid (I), its methyl ester (II) and the endo- vinyl analog of neoxanthobilirubinic acid methyl ester ("half bilirubin" methyl ester) (III) bind to human serum albumin (HSA) and thereby exhibit greatly increased fluorescence as well as induced circular dichroism in their long wavelength transitions. As determined from fluorescence studies, the "tightness" of the binding to HSA is, inter alia , inversely related to the inherent solubility of the dipyrromethenone in aqueous buffer. Fast (ps) configurational isomerization ( Z → E ) is the major pathway for decay of the first excited (singlet) states of the HSA-bound dipyrromethenones. A quantum yield of − 0.4 is associated with the Z → E isomerization. Binding to HSA enhances the lifetimes of the less thermodynamically stable E -isomers. The results of these studies of "half bilirubins" allow a clear evaluation of the effects of protein binding upon the photophysics and photochemistry of bilirubin.     

Fatty Acid Binding to Human Serum Albumin in Blood Serum Characterized by EPR Spectroscopy

One of the functions of Human Serum Albumin (HSA) is binding and transport of fatty acids. This ability could be altered by the presence of several blood components such as toxins or peptides – which in turn alters the functionality of the protein. We aim at characterizing HSA and its fatty acid binding in native serum environment. Native ligand binding and deviations from normal function can be monitored by electron paramagnetic resonance (EPR) spectroscopy using spin labeled fatty acids (FAs). Blood serum from healthy individuals is used to examine healthy HSA in its natural physiological conditions at different loading ratios of protein to FAs. Among the EPR spectroscopic parameters (like hyperfine coupling, line shape, rotational correlation time and population of different binding sites) the rotational correlation time is found to differ significantly between binding sites of the protein, especially at loading ratios of four FAs per HSA. Although differences are observed between individual samples, a general trend regarding the dynamics of healthy HSA at different loading ratios could be obtained and compared to a reference of purified commercially available HSA in buffer.     

PHOTOREACTIONS OF MACROCYCLIC DYES BOUND TO HUMAN SERUM ALBUMIN

The photophysical properties of tetrakis(4-sulfonatophenyl)porphyrin (H2TSPP), its tin (IV) complex (SnTSPP), aluminium(III) trisulfonatophthalocyanine (AIPCS), and the corresponding zinc(II) complex (ZnPCS), have been measured in H2O, D2O, and upon binding to human serum albumin (HSA). The triplet excited states of the various macrocyclic dyes generate singlet molecular oxygen, O2(1 delta g) in high quantum yield upon illumination in O2-saturated solution, even in the presence of HSA. The triplet states also abstract an electron from 4-aminophenol, forming the radical anion of the macrocycle. Quenching rate constants and quantum yields have been measured for the various processes in the presence and absence of HSA. It is found that HSA binds all the dyes at nonspecific sites close to the interface in such a manner that the dyes remain accessible to species residing in the solution phase. Dyes that do not possess axial ligands complexed to the central cation (e.g. H2TSPP, ZnPCS) are able to bind also at a deeper, more specific site on the protein where they are protected from species in solution. Under such conditions, triplet quenching by 4-aminophenol is restricted to long-distance electron tunnelling, for which the rate is relatively slow.     

QUANTUM YIELD and EQUILIBRIUM POSITION OF THE CONFIGURATIONAL PHOTOISOMERIZATION OF BILIRUBIN BOUND TO HUMAN SERUM ALBUMIN*

Abstract— At low fluence rates it is possible to observe a photoequilibrium between Z,Z-bilirubin(BR) and its E isomers(collectively called PBR) bound to human serum albumin(HSA).For excitation centered at 465 nm, the fraction of PBR/HSA in the photoequilibrium mixture was observed to be 0.22 + 0.02. The quantum yield for conversion of BR/HSA to PBR/HSA was found to be 0.20 0.02. The equality of the quantum yield value and the fraction of PBR/HSA in the photoequilibrium mixture is consistent with a simple mechanism for the photoisomerization in which optically excited singlet states of BR and PBR convert rapidly and with virtually total efficiency to a common excited intermediate with twisted geometry that subsequently decays to BR and PBR in the ratio 4:1, respectively. Quantum yields for other photoprocesses of BR bound to HSA are much lower than that for configur-ational isomerization. The central role suggested for configurational isomerization in the phototherapy for neonatal hyperbilirubinemia is supported by these observations.     

Energy transfer and fluorescence quenching in complexes of polymethine dyes with human serum albumin

Electronic excitation energy transfer (EET) between molecules of polymethine dyes bound to human serum albumin (HSA) has been established and studied by absorption and fluorescence spectroscopy as well as by fluorescence decay measurements. In this system, excitation of the donor dye molecule leads to fluorescence of the acceptor dye molecule, both bound to HSA, with donor fluorescence quenching by the acceptor. The short distance between the donor and the acceptor (25-28 A) revealed from the Forster model of EET as well as some spectroscopic data show that both molecules are probably located in the same binding domain of HSA. The role of HSA is to bring donor and acceptor molecules together to a distance adequate to achieve EET as well as to increase the donor and acceptor fluorescence quantum yields. Efficient quenching of the intrinsic HSA fluorescence by some polymethine dyes (oxonols) is observed. The experimental results fit well a model for the formation of a weakly fluorescent dye-HSA complex; the quencher in this complex should be located in the immediate vicinity of the HSA fluorophore group (Trp(214)).     

Aniline as a TICT rotor to derive methine fluorogens for biomolecules: A curcuminoid-BF2 compound for lighting up HSA/BSA

The unique structure of fluorescent proteins in which the fluorophore is encapsulated by the protein shell to restrict rotation and emit light inspired the screening of chromophores that selectively bind to biomolecules to generate fluorescence. In this paper, we report a curcuminoid-BF₂-like fluorescent dye N-BF₂ containing 4-dimethylaniline as an electron-donating group. When this dye is combined with HSA or BSA, the fluorescence is enhanced 90/112-fold, and the fluorescence quantum yield increases from <0.001 to 0.16/0.19. Such a large change in fluorescence enhancement is due to the encapsulation of N-BF₂ in the protein cavity by HSA/BSA, which inhibits the intramolecular rotation of the aniline moiety caused by charge transfer after the fluorophore is excited by light. N-BF₂ has fast and strong binding to HSA or BSA and was found to be reversible in solution and intracellularly. Since N-BF₂ also has the ability to target lipid droplets, the complex of N-BF₂/HSA realizes the regulation of reversible lipid droplet staining in cells.     

Effect of ionic liquid on the native and denatured state of a protein covalently attached to a probe: Solvation dynamics study

Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the solvation dynamics of a probe covalently attached to a protein (human serum albumin (HSA)) has been studied using femtosecond up-conversion. For this study, a solvation probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) has been covalently attached to the lone cysteine group (cys-34) of the protein HSA. Addition of 1.5 M RTIL or 6 M GdnHCl causes a red shift of the emission maxima of CPM bound to HSA by 3 nm and 12 nm, respectively. The average solvation time ?τ(s)? decreases from 650 ps (in native HSA) to 260 ps (～2.5 times) in the presence of 1.5 M RTIL and to 60 ps (～11 times) in the presence of 6 M GdnHCl. This is ascribed to unfolding of the protein by RTIL or GdnHCl and therefore making the probe CPM more exposed. When 1.5 M RTIL is added to the protein denatured by 6 M GdnHCl in advance, a further ～5 nm red shift along with further ～2 fold faster solvent relaxation (?τ? ～30 ps) is observed. Our previous fluorescence correlation spectroscopy study [D. K. Sasmal, T. Mondal, S. Sen Mojumdar, A. Choudhury, R. Banerjee, and K. Bhattacharyya, J. Phys. Chem. B 115, 13075 (2011)] suggests that addition of RTIL to the protein denatured by 6 M GdnHCl causes a reduction in hydrodynamic radius (r(h)). It is demonstrated that in the presence of RTIL and GdnHCl, though the protein is structurally more compact, the local environment of CPM is very different from that in the native state.     

Application of liquid pre-column capillary electrophoresis technique to the study of interaction between drug enantiomers and human serum albumin

Based on the chiral separation of several basie drugs, dimetindene, tetryzoline, theodrenaline and verapamil, the liquid pre-colunm capillary electrophoresis (LPC-CE) technique was established. It was used to determine free concentrations of drug enantiomers in mixed solutions with human serum albumin (HSA). To prevent HSA entering the CE chiral separation zone, the mobility differences between HSA and drugs under a specific pH condition were employed in the LPC. Thus, the detection confusion caused by protein was totally avoided. Further study of binding constants determination and protein binding competitions was carried out. The study proves that the LPC technique could be used for complex media, particularly the matrix of protein coexisting with a variety of drugs.     

Binding of glycyrrhizin to human serum and human serum albumin

The binding of glycyrrhizin (GLZ) to human serum and human serum albumin (HSA) was examined by an ultrafiltration technique. Specific and nonspecific bindings were observed in both human serum and HSA. The association constants (K) for the specific bindings were very similar: 1.31 x 10(5) M-1 in human serum and 3.87 x 10(5) M-1 in HSA. The number of binding sites (n) and the linear binding coefficient (phi) in HSA were 1.95 and 3.09 x 10(3) M-1, respectively. When the human serum protein concentration was assumed to be 4.2% (equal to the measured serum albumin concentration), n in human serum was 3.09, which is similar to the n value in HSA, and phi in human serum was 0.71 x 10(3) M-1, which is reasonably close to that for HSA. The binding pattern of GLZ with human serum protein on Sephadex G-200 column chromatography showed that GLZ binds to only the albumin fraction. It was concluded that the GLZ-binding sites in human serum exist mainly on albumin and GLZ binds to specific and nonspecific binding sites at lower and higher concentrations than approximately 2 mM, respectively.     

Effect of protein binding on the disposition of cephalexin and cefazolin in a simultaneous perfusion system of rat liver and kidney

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.     

Structure and properties of licochalcone A-human serum albumin complexes in solution: a spectroscopic, photophysical and computational approach to understand drug-protein interaction

In the present contribution we address the study of the interaction of a flavonoid-derivative licochalcone A (LA) with human serum albumin (HSA). The application of circular dichroism, UV-Vis absorption, fluorescence and laser flash photolysis combined with molecular mechanics, molecular dynamics and quantum mechanical calculations of rotational strength afforded a clear picture of the modes of association of the LA neutral molecule to HSA, evidencing specific interactions with protein amino acids and their photophysical consequences. The drug is primarily associated in subdomain IIA where a strong interaction with Trp214 is established. At least two different positions of LA with respect to tryptophan are possible, one with the phenolic ring of the drug facing the aromatic ring of Trp214 and the other with the methoxyphenolic ring of LA in proximity to Trp214. In both cases LA is at ca. 4 angstroms from Trp214. This vicinity does not affect much the S1 singlet state deactivation of the bound drug, which exhibits a slightly higher fluorescence quantum yield and fluorescence lifetime on the order of that of the free molecule. The LA triplet lifetime appears to be somewhat shortened in this site. The secondary binding site is in subdomain IIIA. Here, the carbonyl group of LA experiences a strong H-bond with the OH-phenolic substituent of Tyr411. This interaction reduces substantially the LA molecular degrees of freedom, thereby determining a decrease of both radiative and nonradiative rate constants for decay of the singlet. The overall rigidity of the structure causes a lengthening of the triplet lifetime.     

Adsorption and interaction of fibronectin and human serum albumin at the liquid-liquid interface

The goal of this work was to investigate the dynamics of human plasma fibronectin (HFN) at the oil-water interface and to characterize its interactions with human serum albumin (HSA) by total internal reflection fluorescence microscopy (TIRFM). Among key results, we observed that fibronectin adsorption at the oil-water interface is rapid and essentially irreversible, even over short time scales. This may be due to the highly flexible nature of the protein, which allows its various domains to quickly attain energetically favorable conformations. On the other hand, HSA adsorption at the oil-water interface is relatively reversible at short times, and the protein is readily displaced by fibronectin even after HSA has been adsorbed at the interface for as long as 2 h. At longer adsorption times, HSA is able to more effectively resist complete displacement by fibronectin, although we observed significant fibronectin adsorption even under those conditions. Displacement of adsorbed fibronectin by HSA was negligible under all conditions. Fibronectin also adsorbs preferentially from a mixture of HFN and HSA, even when the concentration of HSA is substantially higher. This study is relevant to such emerging research thrusts as the development of biomimetic interfaces for a variety of applications, where there is a clear need for better understanding of the effects of interfacial competition, adsorption time scales, and extent of adsorption irreversibility on interfacial dynamics.     

Denaturation of human serum albumin under the action of cetyltrimethylammonium bromide according to fluorescence polarization data of protein

Denaturation of human serum albumin (HSA) under the action of cationic detergent cetyltrimethylammonium bromide (CTAB) is studied at different pH values by estimating the rotational diffusion of protein via fluorescence polarization. The degree of polarization of HSA tryptophan fluorescence, the rotational relaxation time, the rotational diffusion coefficient and the effective Einstein radius of the HSA molecules in solutions with different CTAB concentrations at different pH values are determined. The obtained rotational diffusion parameters of the HSA molecules show that under the action of CTAB, HSA denaturation has a one-stage character and proceeds more intensely and effectively at pH values higher than the pI value of protein (4.7).     

Microdialysis-liquid chromatographic study on competitive binding of drugs to protein

A new method to determine the interaction between drug and protein has been developed by utilizing the technique of microdialysis sampling with the ketoprofen and the human serum albumin (HSA) as the model of drug and protein.Two kinds of binding sites of HSA to ketoprofen have been observed.The binding constants and number of binding sites obtained by the Scatchard equation are 0.799,3.18×106 mol-1 L and 2.15,2.01×105 mol-1 L,respectively The displacement binding of drugs to HSA has also been studied.The strong displacement of competitive binding of ibuprofen with ketoprofen to HSA was observed,which means that the primary binding site of HSA to ketoprofen and that to ibuprofen are the same.However,only a weaker displacement of warfarin for the association of ketoprofen with HSA was observed,which may suggest that the primary binding site of HSA to ketoprofen is different from that to warfarin.Such a displacement effect for competitive binding of drugs to HSA was explained by the displacement model i     

Osmotic Pressure, Small-Angle X-Ray, and Dynamic Light Scattering Studies of Human Serum Albumin in Aqueous Solutions

Osmotic pressure measurements of human serum albumin (HSA) dissolved in water and in 0.01, 0.1, and 1.0 M phosphate buffer are reported as a function of the protein concentration. Two different forms of the protein were studied: defatted HSA (HSA1) and HSA with fatty acids (HSA2). The measured values of the osmotic coefficient were well below 1, indicating large deviations from ideality even for dilute protein solutions. The measured values increased with increasing HSA concentration and the increase was a function of pH. For higher concentrations of added phosphate buffer, the pH of solution had less influence on the measured osmotic pressure. The osmotic pressure of HSA1 in water was found to be considerably lower than that of the HSA2 modification. This effect was ascribed to formation of dimers in the HSA1 solution. The osmotic measurements were complemented by the small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) studies of dilute HSA solutions in water. The SAXS and DLS data confirmed the dimerization of HSA1 molecules under these conditions. Detailed analysis of the SAXS data suggested a parallel orientation of two protein molecules in a dimer. Copyright 2001 Academic Press.     

PHOTOKINETIC AND PHOTOPHYSICAL MEASUREMENTS OF THE SENSITIZED PHOTOOXIDATION OF THE TRYPTOPHYL RESIDUE IN N-ACETYL TRYPTOPHANAMIDE AND IN HUMAN SERUM ALBUMIN

Abstract— The photosensitized oxidation of 10–100 μ M N -acetyl-L-tryptophanamide (NATA) in neutral aqueous solution and in the presence of various dyes proceeds by a pure O₂(¹Δ_g)-involving mechanism. Incorporation of the tryptophyl (Trp) residue into the polypeptide chain of human serum albumin (HSA) has no influence on the mechanism and efficiency of Trp photooxidation when sensitized either by methylene blue, a non-binding dye, or by rose bengal, a dye that gives non-covalent 1: 1 complexes with HSA. This is due to the location of the Trp residue in close proximity of the protein surface and, in the case of rose bengal, to the coincidence of the photophysical properties (including the quantum yield of O₂(¹Δ_g) generation) for the free and HSA-bound dye. Hematoporphyrin also binds to HSA with 1: 1 stoichiometry, although at a different site from rose bengal. Bound Hp again displays photophysical properties very similar with those of free Hp; however, the efficiency of Trp photo-oxidation in HSA is about 5-fold higher than in NATA owing to a limited rearrangement of the protein structure, induced by Hp binding, which enhances the probability of chemical quenching of O₂(¹Δ_g) by the indole ring.