PEGylation of bovine serum albumin using click chemistry for the application as drug carriers

Monomethyl poly(ethylene glycol) (mPEG)-modified bovine serum albumin (BSA) conjugates (BSA-mPEG) were obtained by the mild Cu(I)-mediated cycloaddition reaction of azided BSA (BSA-N(3) ) and alkyne-terminated mPEG. The structure and characteristics of BSA-mPEG conjugates were thoroughly investigated. There were about two PEG chains conjugated onto each BSA molecule as determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) analysis. The intrinsic nonspecific binding ability of BSA was used for adsorption and sustained release of both rifampicn and 5-fluorouracil (5-FU). The helical structures of BSA were preserved to a large extent after modification and drug adsorption on BSA was confirmed via circular dichroism spectroscopy. Drugs adsorbed onto the conjugated formulation to a lesser extent than on BSA due to mPEG modification. The in vitro release of both rifampicin and 5-FU, however, indicated that BSA-mPEG can function as a drug carrier. Overall, the click reaction provided a convenient tool for the pegylation of BSA. The biological activity of the BSA-mPEG conjugates, including the drug transportation capacity and biocompatibility, were largely retained.     

Synthesis of F16 conjugated with 5‐fluorouracil and biophysical investigation of its interaction with bovine serum albumin by a spectroscopic and molecular modeling approach

5‐Fluorouracil (5‐FU) has been widely used as a chemotherapy agent in the treatment of many types of solid tumors. Investigation of its antimetabolites led to the development of an entire class of fluorinated pyrimidines. However, the toxicity profile associated with 5‐FU is significant and includes diarrhea, mucositis, hand–foot syndrome and myelosuppression. In aiming at reducing of the side effects of 5‐FU, we have designed and synthesized delocalized lipophilic cations (DLCs) as a vehicle for the delivery of 5‐FU. DLCs accumulate selectively in the mitochondria of cancer cells because of the high mitochondrial transmembrane potential (ΔΨ_m). Many DLCs exhibited anti‐cancer efficacy and were explored as potential anti‐cancer drugs based on their selective accumulation in the mitochondria of cancer cells. F16, the DLC we used as a vehicle, is a small molecule that selectively inhibits tumor cell growth and dissipates mitochondrial membrane potential. The binding of the conjugate F16–5‐FU to bovine serum albumin (BSA) was investigated using spectroscopic and molecular modeling approaches. Fluorescence quenching constants were determined using the Stern–Volmer equation to provide a measure of the binding affinity between F16–5‐FU and BSA. The activation energy of the interaction between F16–5‐FU and BSA was calculated and the unusually high value was discussed in terms of the special structural block indicated by the molecular modeling approach. Molecular modeling showed that F16–5‐FU binds to human serum albumin in site II, which is consistent with the results of site‐competitive replacement experiments. It is suggested that hydrophobic and polar forces played important roles in the binding reaction, in accordance with the results of thermodynamic experiments. Copyright © 2012 John Wiley & Sons, Ltd.     

Competition of cytarabine and aspirin in binding to serum albumin in multidrug therapy

The aim of this study was to describe a competition between cytarabine (araC) and aspirin (ASA) in binding with bovine serum albumin (BSA). High-affinity binding sites for both drugs were determined using a spectrofluorimetric method. Cytarabine as well as aspirin binds in the IIA hydrophobic subdomain of the transporting protein. Binding constants for araC-BSA and ASA-BSA were calculated by the Scatchard method. Analysis of ultraviolet (UV) difference spectra showed that araC, which has a higher affinity to BSA in comparison to ASA [Ka(araC) > Ka(ASA)] displaces ASA in high-affinity binding sites.The competition between drugs in low-affinity binding sites was investigated using (1)H nuclear magnetic resonance (NMR) and 13C-NMR spectra. We concluded that in the low-affinity binding sites cytarabine decreases the affinity of albumin toward aspirin. However, the interaction between araC and BSA becomes more difficult in the presence of aspirin.     

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.     

Combined spectroscopies and molecular docking approach to characterizing the binding interaction between lisinopril and bovine serum albumin

To further understand the mode of action and pharmacokinetics of lisinopril, the binding interaction of lisinopril with bovine serum albumin (BSA) under imitated physiological conditions (pH 7.4) was investigated using fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) and molecular docking methods. The results showed that the fluorescence quenching of BSA near 338 nm resulted from the formation of a lisinopril–BSA complex. The number of binding sites (n) for lisinopril binding on subdomain IIIA (site II) of BSA and the binding constant were ~ 1 and 2.04 × 10⁴ M^–1, respectively, at 310 K. The binding of lisinopril to BSA induced a slight change in the conformation of BSA, which retained its α‐helical structure. However, the binding of lisinopril with BSA was spontaneous and the main interaction forces involved were van der Waal's force and hydrogen bonding interaction as shown by the negative values of ΔG⁰, ΔH⁰ and ΔS⁰ for the binding of lisinopril with BSA. It was concluded from the molecular docking results that the flexibility of lisinopril also played an important role in increasing the stability of the lisinopril–BSA complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescence spectrometric study on the interaction of tamibarotene with bovine serum albumin

The interaction between tamibarotene and bovine serum albumin (BSA) was studied using fluorescence quenching technique and ultraviolet–visible spectrophotometry. The results of experiments showed that tamibarotene could strongly quench the intrinsic fluorescence of BSA by a dynamic quenching mechanism. The apparent binding constant, number of binding site and corresponding thermodynamic parameters at different temperatures were calculated respectively, and the main interaction force between tamibarotene and BSA was proved to be hydrophobic force. Synchronous fluorescence spectra showed that tamibarotene changed the molecular conformation of BSA. When BSA concentration was 1.00 × 10^?6 mol L^?1, the quenched fluorescence ΔF had a good linear relationship with the concentration of tamibarotene in the range 1.00 × 10^?6 to 12.00 × 10^?6 mol L^?1 with the detection limit of 6.52 × 10^?7 mol L^?1. Copyright © 2010 John Wiley & Sons, Ltd.     

Flavonoid binding to human serum albumin

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3 × 10⁻⁶ and 3.9 × 10⁻⁵ M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.     

Interactions between m‐phenylenediamine and bovine serum albumin measured by spectroscopy

This study explored interactions between m‐phenylenediamine (MPD) and bovine serum albumin (BSA) by spectrophotometry. The Stern‐Volmer equation and UV‐vis spectra examination at different temperatures and pH were used to explore different quenching mechanisms. Under simulated physiological conditions, the binding distance between MPD and BSA was 5.18 nm with a ratio of 1:1. The quenching effect of MPD on BSA intrinsic fluorescence depended strongly on pH, and maximum quenching was observed at alkaline pH. Moreover, the thermodynamic parameters of the MPD‐BSA system showed that the predominant acting force between MPD and BSA was a hydrophobic force. The impact of MPD on the conformation of BSA and the effects of co‐ions on binding interactions were also examined. Copyright © 2012 John Wiley & Sons, Ltd.     

Studies of phenytoin binding to human serum albumin by high-performance affinity chromatography

High-performance affinity chromatography was used to study the binding of phenytoin to an immobilized human serum albumin (HSA) column. This was accomplished through frontal analysis and competitive binding zonal elution experiments, the latter of which used four probe compounds for the major and minor binding sites of HSA injected into the presence of mobile phases containing known concentrations of phenytoin. It was found that phenytoin can interact with HSA at the warfarin-azapropazone, indole-benzodiazepine, tamoxifen, and digitoxin sites of this protein. The association constants for phenytoin at the indole-benzodiazepine and digitoxin sites were determined to be 1.04 (+/-0.05) x 10(4)M(-1) and 6.5 (+/-0.6) x 10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Both allosteric interactions and direct binding for phenytoin appear to take place at the warfarin-azapropazone and tamoxifen sites. This rather complex binding system indicates the importance of identifying the binding regions on HSA for specific drugs as a means for understanding the transport of such substances in blood and in characterizing their potential for drug-drug interactions.     

Mechanism of bovine serum albumin aggregation during ultrafiltration

Protein fouling is a critical problem for ultrafiltration. In this study, we adopted bovine serum albumin (BSA) as a model protein and polysulfone membrane as a typical ultrafiltration membrane. We then investigated the factors of the protein denaturation and aggregation, such as stirring shear stress and intermolecular exchange of disulfide during ultrafiltration, and discussed the BSA fouling mechanism. Fourier transform-infrared analysis revealed that magnetic stirring did not cause any difference in the secondary structural change of BSA gel-like deposits on the ultrafiltration membrane. BSA aggregates were collected from BSA gel-like deposits on the ultrafiltration membrane by centrifugation. Polyacrylamide gel electrophoresis in SDS analysis of BSA aggregates proved that the major binding of the BSA aggregates involved intermolecular disulfhydryl binding and that capping the free thiol group in BSA molecules with cysteine induced a remarkable decrease in the amount of the BSA aggregates during ultrafiltration. We concluded that one of the main factors in the BSA aggregation during ultrafiltration is the intermolecular exchange of disulfide through cysteinyl residue. We also found that the BSA aggregation caused a decrease in alpha-helix from 66% to 50% and an increase in beta-sheet from 20% to 36%, which was presumably because the cysteine residues associated with the intermolecular disulfide bonds had been located in alpha-helices. Copyright John Wiley & Sons, Inc.     

Application of aqueous two-phase partition to the production of homogeneous preparations of fluorescently labelled human serum albumin

The development of a traceable molecular probe was investigated for the monitoring of partition behaviour of biomolecules in aqueous two-phase systems. This work was based upon the selective labelling of the free thiol group of human serum albumin (i.e. Cys34) with the fluorophore N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid. The preparation of homogeneously labelled protein required purification operations. A succession of five processes was successfully applied, comprising two size-exclusion chromatographic operations by gel filtration and a series of three appropriately manipulated aqueous two-phase systems comprising PEG 1450 and phosphate salt. Aqueous two-phase partitioning is herein presented as an alternative to difficult separation and could be applied for ‘fine’ purifications.     

Variable responsiveness of rat tracheal epithelial cells to bovine serum albumin in serum-free culture

Summary The colony-forming efficiency of rat tracheal epithelial (RTE) cells was determined in serum-free media containing different types of commercially available bovine serum albumin (BSA): crude fraction V, essentially globulin-free, essentially fatty-acid-free, and essentially globulin- and fatty-acid-free BSA. RTE cells exhibited a concentration-dependent increase in colony-forming efficiency in response to crude fraction V BSA. Similar results were obtained using essentially globulin-free BSA. However, deletion of cholera toxin from the medium resulted in a decrease in the colony-forming efficiency for cells plated in high concentrations (>2 mg/ml) of globulin-free, but not one type of fraction V, BSA. Essentially fatty-acid-free or essentially fatty-acid- and globulin-free BSA stimulated RTE cell colony formation at low concentrations (less than 2.5 to 5 mg BSA/ml) but resulted in concentration-dependent decreases in colony-forming efficiency at higher concentrations. The response of cells to these BSAs was not dependent on cholera toxin. Finally, commerically available fraction V BSA prepared by heat shock, dialysis, charcoal treatment, and deionization was stimulatory at low concentrations but inhibitory at high concentrations. These data suggest that impure preparations of BSA can, under different conditions, stimulate or inhibit cell proliferation and that the expression, of these activities is affected by the method of BSA preparation, the concentration of BSA used, and, in some cases, by the presence or absence of cholera toxin. Research conducted with support from the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, under contract no. DE-AC04-76EV01013 in facilities fully acredited by the American Association for Laboratory Animal Care.     

Unveiling the binding mode of perfluorooctanoic acid to human serum albumin

Perfluorooctanoic acid (PFOA) is a toxic compound that is absorbed and distributed throughout the body by noncovalent binding to serum proteins such as human serum albumin (hSA). Though the interaction between PFOA and hSA has been already assessed using various analytical techniques, a high resolution and detailed analysis of the binding mode is still lacking. We report here the crystal structure of hSA in complex with PFOA and a medium‐chain saturated fatty acid (FA). A total of eight distinct binding sites, four occupied by PFOAs and four by FAs, have been identified. In solution binding studies confirmed the 4:1 PFOA‐hSA stoichiometry and revealed the presence of one high and three low affinity binding sites. Competition experiments with known hSA‐binding drugs allowed locating the high affinity binding site in sub‐domain IIIA. The elucidation of the molecular basis of the interaction between PFOA and hSA might provide not only a better assessment of the absorption and elimination mechanisms of these compounds in vivo but also have implications for the development of novel molecular receptors for diagnostic and biotechnological applications.     

Interaction of allylisothiocyanate with bovine serum albumin

The interaction of allylisothiocyanate with bovine serum albumin was monitored by fluorescence titration. The interaction was weak with an apparent association constant of 2 × 10². The interaction was unaffected in the pH range of 5.0 to 8.3 and by NaCl. However, the addition of dioxane upto 4% increased the value of the association constant. N-Methyl bovine serum albumin and bovine serum albumin with sulphydryl groups blocked had the same affinity for allylisothiocyanate suggesting that amino and sulphydryl groups may not be involved in the interaction. Polyacrylamide gel electrophoresis and estimation of available lysine suggested that there were perhaps two types of groups involved in the interaction of allylisothiocyanate with bovine serum albumin. An erratum to this article is available at .     

Structural studies of bovine,equine, and leporine serum albumin complexes with naproxen

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein‐ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA‐NPS), equine (ESA‐NPS), and leporine (LSA‐NPS) determined to 2.58 Å (C2), 2.42 Å (P6₁), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA‐NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA‐NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein. Proteins 2014; 82:2199–2208. © 2014 Wiley Periodicals, Inc.     

Combined spectroscopies and molecular docking approach to characterizing the binding interaction of enalapril with bovine serum albumin

The binding interaction between bovine serum albumin (BSA) and enalapril (ENPL) at the imitated physiological conditions (pH = 7.4) was investigated using UV–vis absorption spectroscopy (UV–vis), fluorescence emission spectroscopy (FES), synchronous fluorescence spectroscopy (SFS), Fourier transform infrared spectroscopy (FT‐IR), circular dichroism (CD) and molecular docking methods. It can be deduced from the experimental results from the steady‐state fluorescence spectroscopic titration that the intrinsic BSA fluorescence quenching mechanism induced by ENPL is static quenching, based on the decrease in the BSA quenching constants in the presence of ENPL with increase in temperature and BSA quenching rates >10¹⁰ L mol^?1 sec^?1. This result indicates that the ENPL–BSA complex is formed through an intermolecular interaction of ENPL with BSA. The main bonding forces for interaction of BSA and ENPL are van der Waal's forces and hydrogen bonding interaction based on negative values of Gibbs free energy change (ΔG ⁰), enthalpic change (ΔH ⁰) and entropic change (ΔS ⁰). The binding of ENPL with BSA is an enthalpy‐driven process due to |ΔH °| > |T ΔS °| in the binding process. The results of competitive binding experiments and molecular docking confirm that ENPL binds in BSA sub‐domain IIA (site I) and results in a slight change in BSA conformation, but BSA still retains its α‐helical secondary structure.     

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.     

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van’t Hoff plots. An enthalpy–entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy–Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.     

Probing the behavior of bovine serum albumin upon binding to atenolol: insights from spectroscopic and molecular docking approaches

Molecular interaction of atenolol, a selective β₁ receptor antagonist with the major carrier protein, bovine serum albumin (BSA), was investigated under imitated physiological conditions (pH 7.4) by means of fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and molecular modeling studies. The steady-state fluorescence spectra manifested that static type, due to formation of the atenolol-BSA complex, was the dominant mechanism for fluorescence quenching. The characteristic information about the binding interaction of atenolol with BSA in terms of binding constant (K_b) were determined by the UV–vis absorption titration, and were found to be in the order of 10³ M^?1 at different temperatures, indicating the existence of a weak binding in this system. Thermodynamic analysis revealed that the binding process was primarily mediated by van der Waals force and hydrogen bonds due to the negative sign for enthalpy change (ΔH⁰), entropy change (ΔS⁰). The molecular docking results elucidated that atenolol preferred binding on the site II of BSA according to the findings observed in competitive binding experiments. Moreover, via alterations in synchronous fluorescence, three-dimensional fluorescence and FT-IR spectral properties, it was concluded that atenolol could arouse slight configurational and micro-environmental changes of BSA.