Binding of porphyrin to human serum albumin. Structure-activity relationships.

The equilibrium binding of hydroxyethyl vinyl deuteroporphyrin (HVD) and of irreversible porphyrin aggregates to human serum albumin was studied at the molecular level. This protein may function as an endogenous drug carrier for porphyrins in photodynamic therapy of tumours. HVD-protein binding studies revealed two types of binding sites, which are attributed to the two HVD isomers. The binding constant for the high-affinity isomer, 2.1 (+/- 0.3) x 10(8) M-1, is similar to that previously determined for protoporphyrin. At the same time the binding constant for the lower-affinity HVD isomer, 1.8(+/- 0.3) x 10(6) M-1, is similar to that previously determined for haematoporphyrin. Irreversible porphyrin aggregates were purified from the haematoporphyrin derivative and from Photofrin and are defined by spectral and chromatographic data. Gel-exclusion studies indicate that the dominant size of these aggregates is ten porphyrin monomeric units. The protein-binding constant of these aggregates is 1.7(+/- 0.2) x 10(5) M-1, with four binding sites per protein molecule. The distinction between the HVD isomers along the porphyrin-protein affinity sequence gives insight into the relationship of porphyrin structure to porphyrin-albumin binding. On the basis of this study an evaluation of human serum albumin as an endogenous carrier for porphyrins (at various aggregation states) in photodynamic therapy of tumours is presented.     

Binding of zinc and cadmium to human serum albumin

1. The interaction of zinc and cadmium ion with human serum albumin (HSA) is evaluated and compared by potentiometric titration method and computer simulation of complex equilibria. 2. Zinc binds to histidine and free amino groups, cadmium in addition to basic functional groups of the protein. 3. Whereas zinc binds stronger in 1:1 complexes, chelate binding favours cadmium ions. 4. Within biological pH-conditions, high amounts Zn(II) and even more of Cd(II) will be bound to HSA.     

Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions

This study directly examines the enthalpic contributions to binding in aqueous solution of closely related anesthetic haloethers (desflurane, isoflurane, enflurane, and sevoflurane), a haloalkane (halothane), and an intravenous anesthetic (propofol) to bovine and human serum albumin (BSA and HSA) using isothermal titration calorimetry. Binding to serum albumin is exothermic, yielding enthalpies (DeltaH(obs)) of -3 to -6 kcal/mol for BSA with a rank order of apparent equilibrium association constants (K(a) values): desflurane > isoflurane approximately enflurane > halothane >or= sevoflurane, with the differences being largely ascribed to entropic contributions. Competition experiments indicate that volatile anesthetics, at low concentrations, share the same sites in albumin previously identified in crystallographic and photo-cross-linking studies. The magnitude of the observed DeltaH increased linearly with increased reaction temperature, reflecting negative changes in heat capacities (DeltaC(p)). These -DeltaC(p) values significantly exceed those calculated for burial of each anesthetic in a hydrophobic pocket. The enhanced stabilities of the albumin/anesthetic complexes and -DeltaC(p) are consistent with favorable solvent rearrangements that promote binding. This idea is supported by substitution of D(2)O for H(2)O that significantly reduces the favorable binding enthalpy observed for desflurane and isoflurane, with an opposing increase of DeltaS(obs). From these results, we infer that solvent restructuring, resulting from release of water weakly bound to anesthetic and anesthetic-binding sites, is a dominant and favorable contributor to the enthalpy and entropy of binding to proteins.     

Binding of the bioactive component jatrorrhizine to human serum albumin

The interaction between Jatrorrhizine with human serum albumin (HSA) were studied by fluorescence quenching technique, circular dichroism (CD) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants (K) are 7.278 x 10(4), 6.526 x 10(4), and 5.965 x 10(4) L.mol(-1) at 296, 303, and 310 K, respectively. The CD spectra and FT-IR spectra have proved that the protein secondary structure changed in the presence of Jatrorrhizine in aqueous solution. The effect of common ions on the binding constants was also investigated. In addition, the thermodynamic functions standard enthalpy (DeltaH(0)) and standard entropy (DeltaS(0)) for the reaction were calculated to be -10.891 kJ.mol(-1) and 56.267 J.mol(-1) K(-1), according to the van't Hoff equation. These data indicated that hydrophobic and electrostatic interactions played a major role in the binding of Jatrorrhizine to HSA. Furthermore, the displacement experiments indicated that Jatrorrhizine could bind to the site I of HSA, which was also in agreement with the result of the molecular modeling study.     

Binding of a spin-labelled photoallergen to human serum albumin

The binding site for 3,3',4',5-tetrachlorosalicylanilide (T4CS), a potent photoallergen, on human serum albumin (HSA) was studied by electron spin resonance spectroscopy using a spin-labelled analogue 3,5-dichlorosalicylamido-4-(2,2,6,6-tetramethylpiperidine 1-oxyl) (DCS-TEMPO) of T4CS in the absence of ultraviolet irradiation. DCS-TEMPO bound non-covalently (K = 5.8 X 10(6) M-1) to one major binding site on HSA. This binding site could be blocked by the photochemical binding of T4CS to the protein. Limited tryptic digestion of HSA or chemical modification of its single tryptophan residue with 2-hydroxy-5-nitrobenzyl bromide was found to reduce the binding constant of the T4CS/DCS-TEMPO-binding site. These observations are in good agreement with earlier conclusions on the nature of the T4CS-binding site and suggest a location for this site close to the single tryptophan residue of the HSA molecule.     

High affinity binding of paclitaxel to human serum albumin.

Paclitaxel, a very potent antitumor agent is a hydrophobic molecule with low aqueous solubility. Its currently used formula (Taxol) contains the drug in a 1 : 1 (v/v) mixture of ethanol and Cremophor EL. To minimize vehicle-related toxicity, we developed a novel, water-soluble formulation in which paclitaxel is bound noncovalently to human serum albumin. For this purpose, studies of the paclitaxel-albumin binding equilibrium were performed. Paclitaxel dissolved in ethanol was added to the aqueous solution of human serum albumin. Precipitated paclitaxel was removed and unbound drug was separated by ultrafiltration. Paclitaxel concentration was measured by RP-HPLC. Binding data were evaluated based both on the Scatchard plot and the general binding equation describing binding equilibria with the stepwise stoichiometric binding constants. The Scatchard plot was found to be curvilinear with a slight positive slope of the final part. Parameters of high affinity specific binding were determined from the initial part of the curve (nsp = 1.3 and Ksp = 1.7 x 10(6) M(-1)). Stoichiometric binding constants were estimated by fitting the general binding equation to the experimental data (K1 = 2.4 x 10(6) M(-1) and K2 = 1.0 x 10(5) M(-1)). Saturation of the protein with paclitaxel, similarly to other ligands of albumin, could not be reached. The greatest observed value of r (number of paclitaxel molecules bound to one albumin molecule) was 6.6.     

Binding of hypocrellin B to human serum albumin and photo-induced interactions

Molecular binding of hypocrellins to human serum albumin (HSA) needs to be further clarified considering the phototherapeutic potentials of hypocrellins to vascular diseases. In the current work, it was estimated that the binding constant of hypocrellin B (HB) to HSA was 2.28 x 10(4) M(-1). Furthermore, based on the fluorescence responses for both HB and the tryptophan of HSA, it was suggested that the binding of HB to HSA should be more specific rather than distributed randomly on the surface of HSA, which was also confirmed by photobleaching of the tryptophan via photosensitization of HB. Besides, it was found that both of the photo-bleaching of the tryptophan and the photo-oxidation of HB were principally oxygen-dependent, suggesting reactive oxygen species generated via the photosensitization of HB, instead of the free radicals of the photosensitizer (HB*-), play the most important role in photodynamic processes.     

Binding of ring-substituted indole-3-acetic acids to human serum albumin

The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.     

Binding of amifostine to human serum albumin: A biophysical study

The aim of this present work is to investigate the interaction between amifostine and human serum albumin (HSA) in simulated physiological conditions by spectroscopic methods to reveal potential toxic effects of the drug. The results reflected that amifostine caused fluorescence quenching of HSA through a static quenching process, which was further confirmed by the electrochemical experiments. The binding constants at 290, 297 and 304 K were obtained as 2.53 × 10⁵/M, 8.13 × 10⁴/M and 3.59 × 10⁴/M, respectively. There may be one binding site of amifostine on HSA. The thermodynamic parameters indicated that the interaction between amifostine and HSA was driven mainly by hydrogen bonding and electrostatic forces. Synchronous fluorescence spectra, circular dichroism and Fourier transform infrared spectroscopy results showed amifostine binding slightly changed the conformation of HSA with secondary structural content changes. Förster resonance energy transfer study revealed high possibility of energy transfer with amifostine‐Trp‐214 distance of 3.48 nm. The results of the present study may provide valuable information for studying the distribution, toxicological and pharmacological mechanisms of amifostine in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

Crystal structure of human serum albumin at 2.5 A resolution.

A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.     

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.     

Binding of isofraxidin to bovine serum albumin

The binding of isofraxidin to bovine serum albumin (BSA) was studied under physiological conditions with BSA concentration of 1.5 x 10(-6) mol x L(-1) and drug concentration in the range of 1.67 x 10(-6) mol x L(-1) to 2.0 x 10(-5) mol x L(-1). Fluorescence quenching spectra in combination with uv absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and CD spectroscopy was used to determine the drug-binding mode, binding constant, and the protein structure changes in the presence of isofraxidin in aqueous solution. The linearity of Scatchard plot indicates that isofraxidin binds to a single class of binding sites on BSA and the values given for the binding constants agree very closely with those obtained by the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated to be -17.63 kJ x mol(-1) and 51.38 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to BSA.     

Binding of anandamide to bovine serum albumin

The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0 degrees C, 10 degrees C, 23 degrees C, and 37 degrees C. The equilibrium dissociation constant (Kd) increases with temperature from 6.87 +/- 0.53 nM at 0 degrees C to 54.92 +/- 1.91 nM at 37 degrees C. Regression analyses of the data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (DeltaG0) is calculated to -43.05 kJ mol-1 with a large enthalpy (DeltaH0) contribution of -42.09 kJ mol-1. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport in aqueous compartments.     

High resolution crystal structures of human Rab4a in its active and inactive conformations

The Ras-related human GTPase Rab4a is involved in the regulation of endocytosis through the sorting and recycling of early endosomes. Towards further insight, we have determined the three-dimensional crystal structure of human Rab4a in its GppNHp-bound state to 1.6 Angstroms resolution and in its GDP-bound state to 1.8 Angstroms resolution, respectively. Despite the similarity of the overall structure with other Rab proteins, Rab4a displays significant differences. The structures are discussed with respect to the recently determined structure of human Rab5a and its complex with the Rab5-binding domain of the bivalent effector Rabaptin-5. The Rab4 specific residue His39 modulates the nucleotide binding pocket giving rise to a reduced rate for nucleotide hydrolysis and exchange. In comparison to Rab5, Rab4a has a different GDP-bound conformation within switch 1 region and displays shifts in position and orientation of the hydrophobic triad. The observed differences at the S2-L3-S3 region represent a new example of structural plasticity among Rab proteins and may provide a structural basis to understand the differential binding of similar effector proteins.     

Binding of the Promen fluorescent probe to human serum albumin: a fluorescence spectroscopic study.

The binding of Promen (6-propionyl-2-methoxynapthalene) to human serum albumin (HSA) was measured by fluorescence spectroscopy, finding only one class of binding sites on the protein. Hydrophobic interactions play an important role to stabilize the complex. Attempts were made to characterize its binding site using as competitors warfarin, phenylbutazone and diazepam, which bind in a specific site or region on the HSA. Fluorescence polarization measurements and spectrofluorimetric results suggest that diazepam and Promen bind at different but interacting binding sites on the HSA. The changes in the fluorescence emission of the bound Promen in the presence of these drugs, allow to use Promen to detect unspecific interactions with the site II on the HSA.     

Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching

Genistein is an isoflavone and phytoestrogen that is a potent inhibitor of cell proliferation and angiogenesis. This study was designed to investigate the binding of genistein to human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 6.7 × 10⁻⁶ to 2.0 × 10⁻⁵ mol L⁻¹ and HSA concentration at 1.5 × 10⁻⁶ mol L⁻¹. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the binding mode, the binding constant and the protein structure changes in the presence of genistein in aqueous solution. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching change did significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (ΔH) and the entropy change (ΔS) were calculated to be −22.24 kJ mol⁻¹and 19.60 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which indicated that hydrophobic and electrostatic interactions play the main role in the binding of genistein to HSA.     

Differential halothane binding and effects on serum albumin and myoglobin.

To understand further the weak molecular interactions between inhaled anesthetics and proteins, we studied the character and dynamic consequences of halothane binding to bovine serum albumin (BSA) and myoglobin using photoaffinity labeling and hydrogen-tritium exchange (HX). We find that halothane binds saturably and with submillimolar affinity to BSA, but either nonspecifically or with considerably lower affinity to myoglobin. Titration of halothane binding with guanidine hydrochloride suggested more protection of binding sites from solvent in BSA as compared with myoglobin. Protection factors for slowly exchanging albumin hydrogens are increased in a concentration-dependent manner by up to 27-fold with 10 mM halothane, whereas more rapidly exchanging groups of albumin hydrogens have either unaltered or decreased protection factors. Protection factors for slowly exchanging hydrogens in myoglobin are decreased by halothane, suggesting destabilization through binding to an intermediate or completely unfolded conformer. These results demonstrate the conformation dependence of halothane binding and clear dynamic consequences that correlate with the character of binding in these model proteins. Preferential binding and stabilization of different conformational states may underlie anesthetic-induced protein dysfunction, as well as provide an explanation for heterogeneity of action.