Quantitative studies on competitive ligand binding to bovine serum albumin by use of the spin label 5-doxyl dodecanoic acid.

The binding of the spin label 5-doxyl dodecanoic acid to bovine serum albumin in phosphate buffer at pH 7.4 was studied by electron spin resonance spectroscopy. Free label and label bound to serum albumin could be quantitatively measured and evaluated from the superposition spectra of these two species with no previous separation. The efficiency relative to the spin label as competitors for binding to serum albumin was studied with salicylic acid and some fatty acids of medium length. The results were represented both by the stoichiometric model involving equilibrium constants Ki, by binding isotherms constructed from the Ki values, and by a purely graphical representation of the experimental data points without connection with any special binding model.     

Dynamic analysis of irreversible adsorption of protein on porous polymer resins as studied by pulse injection method

Irreversible adsorption of bovine serum albumin onto porous polymer resins is examined by a pulse injection method. The experimental results are theoretically analyzed by a model based on the assumption that there exist three kinds of binding sites with different binding rates, which are considered to exist in the vicinity of the outer periphery surface, the inner surface of macropore, and the micropore, or center part, of particles. The three kinds of adsorption rates are also evaluated by a batch method and are nearly equal to the corresponding kinetic parameters by this method. The amount of BSA bound irreversibly to the resins is independent of the protein concentration and the flow rate examined, which suggests that protein molecules penetrate into pores of resins and occupy almost all the effective binding sites in the resins. The total amount of bound BSA shows a pH dependence with a maximum near the isoelectric point of BSA, and the amount of BSA bound at the slowest rate is most largely influenced by pH.     

The specific hydraulic conductivity of bovine serum albumin.

Previous studies of extracellular matrix hydraulic conductivity have characterized the flow resistance of glycosaminoglycans, proteoglycans and collagen. This work focuses on serum albumin, present in significant quantities in many connective tissues, but not previously considered for its role in determining connective tissue flow resistance. The specific hydraulic conductivity of bovine serum albumin solutions, as a function of concentration, was calculated from sedimentation and ultrafiltration data available in the literature. A rigid particle hydrodynamic model compared favorably with these results. Experimental measurements on an albumin ultrafiltration cell were in agreement with this model (within experimental error); furthermore, the experimental data confirmed the theoretical prediction that there is no (or negligible) pressure drop through the concentration polarization layer. Use of the hydrodynamic model for albumin specific hydraulic conductivity with literature values for the hindrance of albumin when passing through a glycosaminoglycan (GAG) matrix allows an estimate of the relative importance of the albumin on tissue hydraulic conductivity: in non-cartilaginous tissues with moderate GAG concentrations, tissue levels of albumin can generate flow resistance effects comparable to those of the GAGs, although well less than the flow resistance of these tissues.     

The binding of flavopiridol to blood serum albumin

Flavopiridol is a potent cyclin-dependant kinase (CDK) inhibitor and is in clinical trials for anticancer treatment. A limiting factor in its drug development has been the high dosage required in human clinical trials. The high dosage is suggested to be necessary because of significant flavopiridol binding to human blood serum. Albumin is the major protein component of blood serum and has been suggested as a likely high affinity binding target. We characterized the binding of human serum albumin to flavopiridol using circular dichroism (hereafter CD). Flavopiridol bound to human serum albumin has a diagnostic CD binding peak at 284 nm. The diagnostic CD binding peak was unobservable for flavopiridol with bovine serum albumin, using the same experimental conditions. However, under higher albumin concentrations a small CD signal is observed confirming, flavopiridol binds to bovine serum albumin as well.     

Drug binding to human serum albumin: abridged review of results obtained with high-performance liquid chromatography and circular dichroism

The drug binding to plasma and tissue proteins are fundamental factors in determining the overall pharmacological activity of a drug. Human serum albumin (HSA), together with alpha1-acid glycoprotein (AGP), are the most important plasma proteins, which act as drug carriers, with drug pharmacokinetic implications, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This review focuses on the combination of biochromatography and circular dichroism as an effective approach for the characterization of albumin binding sites and their enantioselectivity. Furthermore, their applications to the study of changes in the binding properties of the protein arising by the reversible or covalent binding of drugs are discussed, and examples of physiological relevance reported. Perspectives of these studies reside in supporting the development of new drugs, which require miniaturization to facilitate the screening of classes of compounds for their binding to the target protein, and a deeper characterization of the mechanisms involved in the molecular recognition processes.     

Inhibition of specific antibody binding to adult male Schistosoma mansoni by adsorbed host serum components

The ability of anti-schistosome antibody to bind to adult male Schistosoma mansoni was studied, using fluoresceinated Staphylococcus aureus to detect specific antigen-antibody interaction at the parasite's surface. Both freshly perfused parasites and parasites which had had their adsorbed host antigens removed by elution were employed in a series of experimental manipulations to ascertain under what conditions specific antibody binding occurs and what conditions or factors are necessary for the parasite to reconstitute its surface so that specific binding is precluded. Neither normal mouse serum nor normal mouse IgG bound in a specific manner to either fresh or eluted worms. Slight binding was noted with immune mouse serum on both fresh and eluted worms, while immune IgG produced weak binding on fresh worms, but strong binding to eluted worms. This strong binding was reduced to the level seen on fresh worms by pre-incubation of the eluted worm in normal IgG prior to incubation in immune IgG and binding was completely negated by pre-incubation in either normal mouse serum or normal mouse serum minus IgG. The binding of immune IgG to eluted worms was not diminished by pre-incubation in mouse albumin, bovine albumin, or fetal bovine serum. These studies demonstrate that adsorbed host serum components can inhibit specific antigen-antibody interaction at the parasite's surface and suggest that a degree of specificity exists in what the parasite adsorbs from the host. These data further suggest that the protective serum factor or factors may include, but are not limited to, host IgG.     

New mathematical approach for the evaluation of drug binding to human serum albumin by high-performance liquid affinity chromatography

A novel mathematical approach for investigation of drug–human serum albumin (HSA) interactions by means of high-performance liquid affinity chromatography is developed. The model is based on the assumption that two types of competitive binding sites exist on the HSA molecule. The widely used single-site binding equation is extended and a proper mathematical analysis is proposed allowing the determination of the major parameters characterizing the multisite binding (cobinding) process. The utility of the new approach is proved by competitive studies on HSA binding of two model drugs, diazepam and diclofenac.     

Enantioselective binding sites on bovine serum albumin to dansyl amino acids.

The enantioselective binding sites on bovine serum albumin were examined by HPLC using 19 racemic 5-N, N-dimethylamino-1-naphthalenesulfonyl derivatives of alpha-amino acids (dansyl amino acids) as chiral probes. On a bovine serum albumin bonded chiral stationary phase, seven L-forms eluted faster than their D-forms, while ten D-forms eluted before their L-forms. It was speculated that either two classes or two different binding sites exist on bovine serum albumin which can be distinguished by N-dansyl-L-proline and N-dansyl-D-norvaline. This was confirmed by fluorometric experiments where non-fluorescent 1-naphthalenesulfonyl derivatives were synthesized and competitive adsorption experiments were performed.     

Analysis of existing models of spin label movement during spin labeling of biological macromolecules

The spin-labeled bovine serum albumin and IgG were studied in search of an experimental approach for comparison of different models of rotational mobility of spin label. These models are: the model of isotropic motion of spin label together with the macromolecule (IM); the model of highly anisotropic motion of spin label (HAM); and the model of slow isotropic motion of label around the binding site (SIML). The experimental spectra were measured on a common X-band ESR spectrometer and on the unique 140 GHZ (lambda = 2 mm) ESR spectrometer under the same conditions. Theoretical spectra were computer-calculated according to Freed's theory. We have found, that the results of temperature-viscosity experiments in X-band are contradictory to the model of IM both for the BSA and IgG species. The models of HAM and SIML for the BSA give identical X-band spectra. The bovine serum albumin spectra in the 2 mm region strongly contradict to the assumptions of the HAM model. Also, the SIML model fails to describe the experimental spectra in terms of isotropic motion of the spin label around the binding site. X-band spectra of IgG can not be explained by the SIML model, while the same spectra in the 2 mm region can not be explained by the HAM model.     

Kinetics of bilirubin oxidase and modeling of an immobilized bilirubin oxidase reactor for bilirubin detoxification

The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin-serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin-serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K(1) = 6 x 10(7)M(-1) and K(2) = 4.5 x 10(6)M(-1), respectively. The rate of total bilirubin degradation in bilirubin-serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8-mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350muM and a serum albumin concentration of 500muM.     

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.     

Determination of binding affinities of retinoids to retinoic acid-binding protein and serum albumin

Binding affinities of retinoic acid and its synthetic analogues to intracellular retinoic acid-binding protein, which is a possible candidate for mediating their biological function, and to serum albumin, the plasma transport protein, were evaluated. A quantitative method involving elimination of interfering serum albumin by immunoprecipitation was developed to measure the binding efficiency of these retinoids, some of which are active in modifying epithelial differentiation and preventing tumorigenesis. Two cyclopentenyl analogues of retinoic acid and 13-cis-retinoic acid showed, like retinoic acid, a binding efficiency of 100% for the cellular binding protein. With the phenyl, dichlorophenyl and trimethylmethoxyphenyl analogues of retinoic acid, the binding efficiency increased as the substituents on the aromatic ring increased; thus the trimethylmethoxyphenyl analogue binds almost as efficiently as retinoic acid itself. However, the trimethylmethoxyphenyl analogue with a sulphur atom on the side chain has a much decreased binding affinity. The correlation noticed between the binding efficiency of these retinoids and their biological activity in differentiation and/or in the control of tumorigenesis particularly enhances the confidence in the present method of determining the relative binding efficiencies. None of the vitamins, hormones and cofactors tested, showed appreciable affinity for the retinoic acid-binding site. Studies on binding of retinoic acid and its analogues to serum albumin indicate that no correlation exists between binding affinity for albumin and their biological potency.     

Evaluation of drug-human serum albumin binding interactions with support vector machine aided online automated docking

MOTIVATION: Human serum albumin (HSA), the most abundant plasma protein is well known for its extraordinary binding capacity for both endogenous and exogenous substances, including a wide range of drugs. Interaction with the two principal binding sites of HSA in subdomain IIA (site 1) and in subdomain IIIA (site 2) controls the free, active concentration of a drug, provides a reservoir for a long duration of action and ultimately affects the ADME (absorption, distribution, metabolism, and excretion) profile. Due to the continuous demand to investigate HSA binding properties of novel drugs, drug candidates and drug-like compounds, a support vector machine (SVM) model was developed that efficiently predicts albumin binding. Our SVM model was integrated to a free, web-based prediction platform (http://albumin.althotas.com). Automated molecular docking calculations for prediction of complex geometry are also integrated into the web service. The platform enables the users (i) to predict if albumin binds the query ligand, (ii) to determine the probable ligand binding site (site 1 or site 2), (iii) to select the albumin X-ray structure which is complexed with the most similar ligand and (iv) to calculate complex geometry using molecular docking calculations. Our SVM model and the potential offered by the combined use of in silico calculation methods and experimental binding data is illustrated.     

Using offset recombinant polymerase chain reaction to identify functional determinants in a common family of bacterial albumin binding domains

The 46 amino acid GA albumin binding module is a putative virulence factor that has been identified in 16 domains from four bacterial species. Aside from their possible effects on pathogenicity and host specificity, the natural genotypic and phenotypic variations that exist among members of this module offer unique opportunities for researchers to identify and explore functional determinants within the well-defined sequence space. We used a recently developed in vitro recombination technique, known as offset recombinant PCR, to shuffle seven homologues that encode a broad range of natural GA polymorphisms. Phage display and selection were applied to probe the recombinant library for members that showed simultaneous improvements to human and guinea pig serum albumin binding. Thermodynamic data for the most common phage-selected mutant suggest that domain-stabilizing mutations substantially improved GA binding for both species of albumin.     

Deuteroporphyrin-albumin binding equilibrium. The effects of porphyrin self-aggregation studied for the human and the bovine proteins.

The binding equilibrium of deuteroporphyrin IX to human serum albumin and to bovine serum albumin was studied, by monitoring protein-induced changes in the porphyrin fluorescence and taking into consideration the self-aggregation of the porphyrin. To have control over the latter, the range of porphyrin concentrations was chosen to maker dimers (non-covalent) the dominant aggregate. Each protein was found to have one high-affinity site for deuteroporphyrin IX monomers, the magnitudes of the equilibrium binding constants (25 degrees C, neutral pH, phosphate-buffered saline) being 4.5 (+/- 1.5) X 10(7) M-1 and 1.7 (+/- 0.2) X 10(6) M-1 for human serum albumin and for bovine serum albumin respectively. Deuteroporphyrin IX dimers were found to bind directly to the protein, each protein binding one dimer, with high affinity. Two models are proposed for the protein-binding of porphyrin monomers and dimers in a porphyrin system having both species: a competitive model, where each protein molecule has only one binding site, which can be occupied by either a monomer or a dimer; a non-competitive model, where each protein molecule has two binding sites, one for monomers and one for dimers. On testing the fit of the data to the models, an argument can be made to favour the non-competitive model, the equilibrium binding constants of the dimers, for the non-competitive model (25 degrees C, neutral pH, phosphate-buffered saline), being: 8.0 (+/- 1.8) X 10(8) M-1 and 1.2 (+/- 0.6) X 10(7) M-1 for human serum albumin and bovine serum albumin respectively.     

Carnosine binding: characterization of steric and charge requirements for ligand recognition.

The steric and charge requirements for binding of l-carnosine (β-alanyl-l-histidine) by bovine serum albumin were investigated with proton magnetic resonance (¹HMR) spectrometry. The histidinyl side chain of the dipeptide is responsible for primary recognition by the binding site. Furthermore, recognition is specific to a particular orientation of the histidinyl side chain that is determined by the other amino acid residue of the dipeptide. It was found that, although salts do not have a great effect on the binding of carnosine to bovine serum albumin, this binding cannot be measured by equilibrium dialysis in the presence of salt because of formation of a complex Donnan equilibrium. Carnosine, which has been postulated to have a role in olfaction, binds to the crude particulate fraction of nasal olfactory epithelium in the same steric orientation as it does to bovine serum albumin. Therefore, we have used the binding of carnosine to bovine serum albumin as a model system to test potential competitive inhibitors of carnosine binding that ultimately could be tested for activity in the olfactory pathway. It was found that the binding of carnosine to bovine serum albumin is a good model of nonspecific binding of carnosine to tissue preparations but not of the specific binding of carnosine to the nasal olfactory epithelium. In addition to requiring the proper conformation of the histidinyl residue, the binding to olfactory epithelium also appears to require recognition of the β-alanyl residue and of substituents on the imidazole ring. Evidence is provided that the carnosine binding by the nasal olfactory epithelium demonstrated by ¹HMR spectroscopy does not occur with the mature olfactory receptor neurons.     

How does displacement of albumin-bound tryptophan cause sustained increases in the free tryptophan concentration in plasma and 5-hydroxytryptamine synthesis in brain?

Models of tryptophan catabolism and binding to serum albumin are presented to explain the observed effect of displacement of tryptophan from albumin on the concentrations of free and bound tryptophan and on the rate of 5-hydroxytryptamine (5-HT) synthesis from tryptophan in the brain. A rapid rate of dissociation of tryptophan from albumin (compared to the transit time of tryptophan through the liver) and a large fractional extraction of the free pool of tryptophan during passage through the liver are shown to be necessary factors in determining the effects observed. Because of the low fractional extraction of free tryptophan in the brain, the synthesis of 5-HT will be dependent only upon the free pool of tryptophan. Dissociation of tryptophan from albumin only causes a sustained increase in 5-HT synthesis in the brain because of the effect that this dissociation has on hepatic tryptophan catabolism and thereby on the free pool of tryptophan.     

Flavonoid aglycones can compete with Ochratoxin A for human serum albumin: a new possible mode of action

The mycotoxin Ochratoxin A (OTA) appears worldwide in cereals, plant products, different foods and drinks. Ochratoxin A binds to plasma albumin with a very high affinity. However, it is well known that natural flavonoids can also bind to human serum albumin (HSA) at the same binding site as OTA does (site I, subdomain IIA). A few experimental literature data suggest that reducing the bound fraction of OTA speeds up its elimination rate with a potential decrease in its toxicity. In our experimental model competitive binding properties of flavonoid aglycones were examined with a fluorescence polarization based approach. Our data show that some of the flavonoids are able to remove the toxin from HSA. We conclude that among the 13 studied flavonoid aglycones galangin and quercetin were the most effective competitors for OTA.     

Determining molecular binding sites on human serum albumin by displacement of oleic acid

An NMR method was developed for determining binding sites of small molecules on human serum albumin (HSA) by competitive displacement of (13)C-labeled oleic acid. This method is based on the observation that in the crystal structure of HSA complexed with oleic acid, two principal drug-binding sites, Sudlow's sites I (warfarin) and II (ibuprofen), are also occupied by fatty acids. In two-dimensional [(1)H,(13)C]heteronuclear single quantum coherence NMR spectra, seven distinct resonances were observed for the (13)C-methyl-labeled oleic acid as a result of its binding to HSA. Resonances corresponding to the major drug-binding sites were identified through competitive displacement of molecules that bind specifically to each site. Thus, binding of molecules to these sites can be followed by their displacement of oleic acids. Furthermore, the amount of bound ligand at each site can be determined from changes in resonance intensities. For molecules containing fluorine, binding results were further validated by direct observations of the bound ligands using (19)F NMR. Identifying the binding sites for drug molecules on HSA can aid in determining the structure-activity relationship of albumin binding and assist in the design of molecules with altered albumin binding.     

Thermodynamic study of taurocholate binding to rat serum albumin

Taurocholate binding to rat serum albumin was studied by equilibrium dialysis. The bile salt-protein interaction was studied under different experimental conditions with respect to temperature; ionic strength; Cl- concentration; pH and the presence of butanol in the medium. The results obtained suggest the existence of two binding sites for taurocholate on the albumin molecule, and indicate that both electrostatic and hydrophobic interaction play a role in the binding process.