Urea‐induced binding between diclofenac sodium and bovine serum albumin: a spectroscopic insight

We investigated the interaction of diclofenac sodium (Dic.Na) with bovine serum albumin (BSA) in the absence and presence of urea using different spectroscopic techniques. A fluorescence quenching study revealed that the Stern–Volmer quenching constant decreases in the presence of urea, decreasing further at higher urea concentrations. The binding constant and number of binding sites were also evaluated for the BSA–Dic.Na interaction system in the absence and presence of urea using a modified Stern–Volmer equation. The binding constant is greater at high urea concentrations, as shown by the fluorescence results. In addition, for the BSA–Dic.Na interaction system, a static quenching mechanism was observed, which was further confirmed using time‐resolved fluorescence spectroscopy. UV–vis spectroscopy provided information about the formation of a complex between BSA and Dic.Na. Circular dichroism was carried out to explain the conformational changes in BSA induced by Dic.Na in the absence and presence of urea. The presence of urea reduced the α‐helical content of BSA as the Dic.Na concentration varied. The distance r between the donor (BSA) and acceptor (Dic.Na) was also obtained in the absence and presence of urea, using fluorescence resonance energy transfer. Copyright © 2015 John Wiley & Sons, Ltd.     

Exploring the differences and similarities between urea and thermally driven denaturation of bovine serum albumin: intermolecular forces and solvation preferences

The interactions of bovine serum albumin (BSA) with urea/water were investigated by computer simulation. It was revealed that the BSA-hydrophobic residues in urea solutions favored contact with urea more than with water. Energy decomposition analysis showed that van der Waals energy was the dominant driving force behind urea affinity for hydrophobic residues, whereas coulombic attraction was largely responsible for water affinity for these residues. Meanwhile, urea–BSA hydrogen bond energies were found to be weaker than water–BSA hydrogen bond energies. The greater strength of water–BSA hydrogen bonds than urea–BSA hydrogen bonds, and the opposing preferential interaction between the BSA and urea suggest that disruption of hydrophobic interaction predominates urea–protein denaturation. In pure water, hydrophobic residues showed aggregation tendencies at 323 K, suggesting an increase in hydrophobicity, while at 353 K the residues were partly denatured due to loss of hydrogen bonds; thus, disruption of hydrophobic interactions appeared to contribute less to thermal denaturation.     

Mechanism of the interactions of aliphatic alcohols with bovine serum albumin in the ternary systems—1H n.m.r. study: 1. Aqueous solutions BSA-alcohols-urea

The molecular mechanism of the interaction of aliphatic alcohols (A) with bovine serum albumin (BSA) protein was studied in aqueous solutions at increasing concentrations (0–8 m) of urea (U). ¹H n.m.r. spectra of alcohols were monitored in D₂O in the control binary systems (A—U) and (A—BSA), and in the ternary systems (A—U—BSA) at pH 7.0. Marked and selective broadening of the n.m.r. lines of alcohols in the system (A—BSA) was reduced upon addition of urea, indicating that alcohols are poorly bound by urea-denaturated BSA. The reduction in the ability to associate with BSA depends on chain position of the alcohol molecule and is much higher for α-methylenes (next to ?OH) than for other proton groups. Besides this reduction seems to be a two-step phenomenon dependent upon urea concentration. The results obtained can be explained by competition in formation by the peptide linkages of a protein of the hydrogen bonds with ?OH group of alcohols or fragments of urea molecules.     

Bromophenol blue binding as a probe to study urea and guanidine hydrochloride denaturation of bovine serum albumin

Urea and guanidine hydrochloride (GdnHCl) denaturation of bovine serum albumin (BSA) were investigated using bromophenol blue (BPB) binding as a probe. Addition of BPB to BSA produced an absorption difference spectrum in the wavelength range, 525-675 nm with a minimum at 587 nm and a maximum at 619 nm. The magnitude of absorption difference (DeltaAbs.) at 619 nm decreased on increasing urea/GdnHCl concentration and followed the denaturation curve. The denaturation was found to be a two-state, single-step transition. The transitions started at 1.75 and 0.875 M and completed at 6.5 and 3.25 M with the mid point occurring around 4.0 and 1.5 M urea and GdnHCl concentrations, respectively. The value of free energy of stabilization, DeltaGDH2O as determined from urea and GdnHCl denaturation curves was found to be 4041 and 4602 cal/mol, respectively. Taken together, these results suggest that BPB binding can be used as a probe to study urea and GdnHCl denaturation of BSA.     

Intermolecular interactions in solutions of serum albumin

The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis we have shown the existence of dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0–1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M) the sizes of aggregates decreased, while higher urea concentrations (2–8 M) induced formation of larger aggregates due to the unfolding of the protein.     

Association of the radiosensitizers metronidazole and misonidazole (RO-07-0582) with bovine serum albumin. Effect of urea and ionic strength

The stability of association of nitroimidazole radiosensitizers (metronidazole and misonidazole) with bovine serum albumin (BSA) was examined in aqueous solutions by 1H n.m.r. spectroscopy in the presence of urea (0-8M) as denaturant, or high salt concentration (NaCl0-5M). A broadening of n.m.r. lines of the two radiosensitizers observed in the presence of BSA disappeared with increasing urea concentration. An especially large narrowing effect was observed for the lines attributed to the methylene group near to the hydroxyl in the side chain of misonidazole. The results suggest a release of both radiosensitizers from their binding sites on unfolding by urea of the polypeptide chain of BSA. The increase of ionic strength I caused a monotonic enhancement of broadening by BSA of the metronidazole lines. For misonidazole, the enhancement of broadening was observed at values of I greater than 1, but at low (less than 1 M) concentrations of NaCl the broadening disappeared. Thus, the results obtained in the systems with salt reflect quantitative changes in hydrophobic and hydrogen-bonded contributions to binding of aliphatic moieties of radiosensitizers to BSA.     

SANS/SAXS study of the BSA solvation properties in aqueous urea solutions via a global fit approach

We report on the solvation properties and intermolecular interactions of a model protein (bovine serum albumine, BSA) in urea aqueous solutions, as obtained by combining small-angle neutron and X-ray scattering experiments. According to a global fit strategy, all the whole set of scattering curves are analysed by considering a unique model which includes the BSA structure, the protein-protein interactions and the thermodynamic exchange process of water/urea molecules at the protein solvent interface. As a main result, the equilibrium constant that accounts for the difference in composition between the bulk solvent and the protein solvation layer is derived. Results confirm that urea preferentially sticks to the protein surface, inducing a noticeable change in both the repulsive and the attractive interaction potentials.     

Use of domain specific ligands to study urea-induced unfolding of bovine serum albumin

Urea-induced structural transitions in different domains of bovine serum albumin (BSA) were studied fluorometrically using domain specific ligands; chloroform, bilirubin, and diazepam. Urea denaturation of BSA showed a two-step, three-state transition with the accumulation of an intermediate around 4.8-5.2 M urea. During first transition (0-5.0 M urea), a continuous decrease (starting from 1.0 M urea) in diazepam (a ligand for domain III) binding and a late (from 3.0 M urea onward) decrease in chloroform (a ligand primarily for domain I) binding suggested major conformational changes in domain III and partial but significant loss of native conformation in domain I prior to intermediate formation. Absence of any decrease in bilirubin (a ligand for domain II) binding up to 4.5 M urea indicated non-involvement of domain II in the unfolding of BSA in this region. However, decrease in bilirubin binding during second transition reflected the unfolding of domain II and its separation from domain I.     

Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.     

Protein stabilizing potential of simulated honey sugar cocktail under various denaturation conditions

Protein stabilizing potential of simulated honey sugar cocktail (SHSC) against chemical and thermal denaturations was studied using bovine serum albumin (BSA) as the model protein. The two-step, three-state transition of urea denaturation of BSA became a single-step, two-state transition along with the shift in the whole transition curve towards higher urea concentrations in the presence of increasing SHSC concentrations [8–20% (w/v)] as revealed by far-UV CD, fluorescence and UV difference spectroscopic results. Far-UV and near-UV CD spectra, UV difference spectra, ANS fluorescence and three-dimensional fluorescence results suggested significant retention of native-like conformation in 4.6 M urea-denatured BSA in the presence of 20% (w/v) SHSC. A significant shift was also noticed in thermal and GdnHCl denaturation curves of BSA in the presence of 20% (w/v) SHSC. Taken together, all these results suggested significant stabilization of BSA against urea, GdnHCl and thermal denaturations by SHSC.     

Reversible denaturation with urea of rabbit liver fructose-1,6-bisphosphatase

Denaturation of fructose-1,6-bisphosphatase (Fru-P2-ase, EC 3.1.3.11) by urea and renaturation of denatured enzyme has been investigated. Denaturation lowers the specific activity of the enzyme but even at 8 M urea concentration in the presence of sucrose the activity of the enzyme is detectable. Centrifugation of the enzyme in a sucrose density gradient at 4 M urea reveals one peak of protein corresponding to a dimer. Denaturation increases intensity of intrinsic fluorescence of Fru-P2-ase and causes a red shift of fluorescence peak of the thioisoindole derivative of the enzyme. Renaturation of the denatured enzyme followed as the reappearance of enzymatic activity in the presence and absence of bovine serum albumin (BSA) is characterised by first order kinetics, k = 1.78 X 10(-3) s-1. The presence of BSA does not affect the rate of renaturation but perceptibly increases the recovery of enzymatic activity. A 100% recovery of Fru-P2-ase activity is observed at 0.5 micrograms/mL concentration of the enzyme and 2 mg/mL of BSA.     

Combining time‐resolved fluorescence with synchronous fluorescence spectroscopy to study bovine serum albumin‐curcumin complex during unfolding and refolding processes

We investigated the complex interaction between bovine serum albumin (BSA) and curcumin by combining time‐resolved fluorescence and synchronous fluorescence spectroscopy. The interaction was significant and sensitive to fluorescence lifetime and synchronous fluorescence characteristics. Binding of curcumin significantly shortened the fluorescence lifetime of BSA with a bi‐molecular quenching rate constant of k_q = 3.17 × 10¹² M^‐1s^‐1. Denaturation by urea unfolded the protein molecule by quenching the fluorescence lifetime of BSA. The tyrosine synchronous fluorescence spectra were blue shifted whereas the position of tryptophan synchronous fluorescence spectra was red shifted during the unfolding process. However, denaturation of urea had little effect on the synchronous fluorescence peak of tyrosine in curcumin‐BSA complex except in the low concentration range; however, it shifted the peak to the red, indicating that curcumin shifted tryptophan moiety to a more polar environment in the unfolded state. Decreases in the time‐resolved fluorescence lifetime and curcumin‐BSA complex during unfolding were recovered during refolding of BSA by a dilution process, suggesting partial reversibility of the unfolding process for both BSA and curcumin‐BSA complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Dielectric study of the urea denaturation of delipidated and relipidated bovine serum albumin

Dielectric relaxation and viscosity measurements were performed on delipidated and relipidated samples of bovine serum albumin (BSA) at urea concentrations between O and 6M. By the combined interpretation of these two hydrodynamic methods the characterization of conformational changes of the molecule during urea denaturation is possible. The denaturation of delipidated BSA results from two mechanisms. The first one is a slow, time-dependent elongation of the molecule; the second one is a rapid swelling which becomes most pronounced at urea concentrations higher than 4M. For relipidated albumin, the slow elongation mechanism occurs but the presence of fatty acids protects the protein aganist molecular swelling. In both cases these conformational changes are accompanied by an increased disymmetry of charge repartition and a concomitant increase of the dipole moment. From these results it follows that lipidated albumin (as occurs under physiological conditions) is less sensitive to denaturation than delipidated albumin.     

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

Curdlan gels as protein drug delivery vehicles

The use of curdlan, a natural -1,3-glucan, in protein drug delivery vehicles was studied by carrying out in vitro release studies with curdlan gels containing bovine serum albumin (BSA) as a model protein. Addition of urea (8 M) decreased the gel formation temperature to 37°C. Curdlan was hydroxyethylated in order to form gels under mild conditions such as physiological temperature and pH. In gels formed in 8 M urea solution, urea was almost released after 2 h while BSA was completely released after 45–100 h. The total time for complete release of BSA increased with curdlan concentration within gels. The strength of hydroxyethylated curdlan gels (385.7 dyne cm^–2) was weaker than that of curdlan gels formed in 8 M urea solution (6277 dyne cm^–2).     

GM1-induced structural changes of bovine serum albumin after chemical and thermal disruption of the secondary structure: a spectroscopic comparison

GM1-induced structural transitions of native and unfolded conformers of bovine serum albumin (BSA) have been studied where in the unfolded conformers, the secondary structures were disrupted either chemically by 8 M urea or thermally by heating at 65 degrees C. With decreasing protein:ganglioside ratio at pH 7.0, the native BSA partially unfolds and expands, while the urea-denatured BSA forms an alpha-helical structural pattern with shrinking in the conformational space. However, a continuous loss of alpha-helicity with minor increase in size was observed for the thermally altered protein in the presence of the GM1 micelle. The changes in the secondary structural content were followed by far-UV circular dichroism (CD) analysis. The dynamic light scattering (DLS) experiments were used to study the variation of the size of the protein-GM1 complexes with increasing concentration of the GM1. Fluorescence experiments show that tryptophan residues of BSA experience a more hydrophobic environment in the presence of the GM1 micelle with a decreasing protein:ganglioside ratio at pH 7.0. The present study shows that GM1 has a strong effect on the conformation of BSA depending on the conformational states of the protein that would relate to a physiological function of GM1 such as acting as the receptor of proteins in the cell membrane.     

Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin.

The non-linearity of the fluorescence emission on increasing the probe to protein ratio has long been regarded as problematic and has lead to the development of dyes to overcome this effect. One of the causes of this non-linear response can be ascribed to the overlap of the label's own absorption and emission spectra. At higher labelling ratios, this affords the possibility of a reasonably efficient energy migration pathway, thus reducing the observed quantum yield of the dye. In this work we study the photophysics of fluorescein isothiocyanate (FITC) when conjugated to bovine serum albumin (BSA) at different labelling ratios (in the range FITC : BSA 1 : 17-15 : 1) using both steady state and time-resolved fluorescence techniques where on going from under labelled to over labelled samples a decrease in the initial (and steady state) anisotropy is observed, accompanied by an increase in the complexity of the decay kinetics and a decrease in the average lifetime. The band structure, elucidated by synchronous scan fluorescence spectroscopy, is also found to change on increased labelling. These results can be applied to the study of protein conformation and were confirmed by the analysis of denaturing BSA using urea.     

Static and dynamic quenching of protein fluorescence. I. Bovine serum albumin.

The fluorescence parameters, lifetime, relative quantum yield, maximum and mean wavelength, half-width, and polarization, of bovine serum albumin (BSA) were measured at 15°C in aqueous solutions containing varying concentrations of different chemical perturbants, glycerol, Cu²⁺ ions, guanidine hydrochloride, and urea. By considering a quenching mechanism as being either dynamic or static, depending upon whether the quenching is or is not accompanied by a change in the fluorescence lifetime, we were able to correlate the changes produced in the various fluorescence parameters by the different chemical perturbants with changes in macromolecular structure as the concentration of perturbant was gradually increased. The addition of glycerol and of Cu²⁺ ions indicated that in aqueous BSA both tryptophan residues are below the surface of the macromolecule, out of contact with solvent water, and, as a consequence, they are statically quenched. “Ultra-Pure” guanidine hydrochloride at 2.4 M or more caused a drastic conformation change, which resulted in the emergence of a visible tyrosine peak at 304 nm in the BSA fluorescence spectrum when either 260- or 270-nm excitation was employed. With the same excitation, the enhancement of BSA tyrosine fluorescence by 6–8 M ultra-pure urea produced only a shoulder near 304 nm in the BSA fluorescence spectrum. We have introduced the use of a new relative quantum yield for protein fluorescence, q′, referenced to the quantum yield of unquenched free tryptophan, which eliminates the quenching action of water from the reference.     

Foam behavior of biological media

Summary The influence of the concentrations of NaCl, NaJ, KJ and/or Na₂SO₄ on the foaminess of BSA solutions is investigated. The foaminess increases with increasing salt concentrations as expected for NaCl, NaJ and Na₂SO₄. With KJ the foaminess exhibits an anomaly. The dependence of the foaminess on the pH is complex. In the presence of buffer there is a minimum at 4.81 and a maximum at 4.7. In the absence of buffer the foaminess reaches a maximum at pH 4 and a minimum at 3. The anomaly of BSA solutions is well-known but not yet fully understood.Symbols BSA Bovine Serum Albumin - C concentration - C_BSA concentration of BSA - C_BUFFER concentration of buffer - C_SALT concentration of salt - V_s equilibrium volume of the foam - V_tg volumetric gas flow rate - =V_s/V_tg foaminess     

Adsorption behavior of bovine serum albumin on lowly activated anionic exchangers suggests a new strategy for solid-phase proteomics

Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.