Hydration in protein folding: thermal unfolding/refolding of human serum albumin

Human serum albumin (HSA) is known to undergo both reversible and irreversible thermal unfolding and refolding, depending upon the experimental conditions (end temperature) at neutral pH. In this report we have used high precision densimetric and ultrasonic measurements to determine the apparent specific volume (phi v) and compressibility (phi k) of HSA at different unfolded and refolded states at two different end temperatures, 55 degrees C and 70 degrees C. The unfolded and refolded states were characterized using dynamic light scattering (DLS), circular dichroism (CD), picosecond-resolved fluorescence decay, and anisotropy of the single-tryptophan residue in HSA (Trp214). Both the unfolded states were allowed to refold by cooling wherein the former and latter processes were found to be reversible and irreversible, respectively, in nature. The results obtained from the densimetric and ultrasonic measurements reveal that the apparent specific volume and compressibility of the protein in the reversible protein unfolding process is preserved upon restoration of HSA to ambient temperature. However, a significant change in phi v and phi k occurs in the process of irreversible protein refolding (from 70 to 20 degrees C). The experimental observation is rationalized in terms of the exposure of domain IIA to an aqueous environment, resulting in the swelling of the protein to a higher hydrodynamic diameter. Our studies attempt to explore the extent of hydration associated with the structural integrity of the popular protein HSA.     

Human serum albumin unfolding pathway upon drug binding: A thermodynamic and spectroscopic description

The interest on phenothiazine drugs has been increased during last years due to their proved utility in the treatment of several diseases and biomolecular processes. In the present work, the binding of the amphiphilic phenothiazines promazine and thioridazine hydrochlorides to the carrier protein human serum albumin (HSA) has been examined by ζ-potential, isothermal titration calorimetry (ITC), fluorescence and circular dichorism (CD) spectroscopies, and dynamic light scattering (DLS) at physiological pH with the aim of analyzing the role of the different interactions in the drug complexation process with this protein. The ζ-potential results were used to check the existence of complexation. This is confirmed by a progressive screening of the protein charge up to a reversal point as a consequence of drug binding. On the other hand, binding causes alterations on the tertiary and secondary structures of the protein, which were observed by fluorescence and CD spectroscopies, involving a two-step, three-state transition. The thermodynamics of the binding process was derived from ITC results. The binding enthalpies were negative, which reveal the existence of electrostatic interactions between protein and drug molecules. In addition, increases in entropy are consistent with the predominance of hydrophobic interactions. Two different classes of binding sites were detected, viz. Binding to the first class of binding sites is dominated by an enthalpic contribution due to electrostatic interactions whereas binding to a second class of binding sites is dominated by hydrophobic bonding. In the light of these results, protein conformational change resembles the acid-induced denaturation of HSA with accumulation of an intermediate state. Binding isotherms were derived from microcalorimetric results by using a theoretical model based on the Langmuir isotherm. On the other hand, the population distribution of the different species in solution and their sizes were determined through dynamic light scattering (DLS). Aggregation of drug/protein complexes was found as a result of a possible expansion of protein structure induced at high drug concentrations. In addition, the presence of free drug aggregates at concentrations below the drug critical micelle concentration was also detected.     

高压CO~2对反胶束溶解蛋白质性质的影响

在308.15K下,研究了表面活性剂琥珀酸二(2-乙基己基)酯磺酸钠(Aerosol-Ot,简称AOT)的浓度和水的含量不同时,溶解的CO~2对反胶束溶解牛血清蛋白(BSA)的性质和异辛烷中AOT反胶束稳定性的影响。实验表明,在适当条件下,CO~2可以使反胶束溶液中的蛋白质全部析出。本研究对有关机理进行了初步分析。     

Studies on the interaction between imidacloprid and human serum albumin: Spectroscopic approach

The interaction between imidacloprid (IMI) and human serum albumin (HSA) was investigated using fluorescence and UV/vis absorption spectroscopy. The experimental results showed that the fluorescence quenching of HSA by IMI was a result of the formation of IMI–HSA complex; static quenching was confirmed to result in the fluorescence quenching. The apparent binding constant K_A between IMI and HSA at three differences were obtained to be 1.51 × 10⁴, 1.58 × 10⁴, and 2.19 × 10⁴ L mol^?1, respectively. The thermodynamic parameters, ΔH° and ΔS° were estimated to be 28.44 kJ mol^?1, 174.76 J mol^?1 K^?1 according to the van’t Hoff equation. Hydrophobic interactions played a major role in stabilizing the complex. The distance r between donor (HSA) and acceptor (IMI) was obtained according to fluorescence resonance energy transfer. The effect of IMI on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy CD and three-dimensional fluorescence spectra, the environment around Trp and Tyr residues were altered.     

Site-selective probe for investigating the asynchronous unfolding of domains in bovine serum albumin

A convenient method is proposed for precise investigation of the asynchronous structural transition of the domains in bovine serum albumin (BSA) during unfolding process. The method is based on a site-selective probe, alizarin red S (ARS), which has a high affinity to the subdomain IIA of BSA. BSA-ARS complex was formed and gradually unfolded by urea from 0 to 8.0 M. The unfolding occurred in different domains of BSA resulted in distinct alterations of the microenvironment of the bound ARS. The spectral response of BSA-ARS complex, including the color, the UV absorption at 530 and 432 nm, and the intrinsic fluorescence at 342 and 310 nm with the excitation wavelength of 280 nm, showed slight changes in the urea concentration from 0 to 4.5 M, drastic changes from 4.5 to 6.0 M, and almost no changes from 6.0 to 8.0 M. The redox behavior of bound ARS between 0.3 and 0.8 V also showed the same trend. Consequently, a two-step, three-state transition process was monitored by naked eyes, UV-vis spectroscopy and electrochemistry. It is the first report to realize the indicator of the intermediate state during the unfolding process of BSA through convenient methods instead of expensive approaches. The work provides a facile method for the investigation of the unfolding process of multidomain proteins.     

Effect of ethanol on the thermal stability of human serum albumin

The effect of ethanol on human serum albumin stability in aqueous solution was studied by use of differential scanning calorimetry. A deconvolution of DSC traces in 2-state model with ΔC _p=0 and ΔC _p≠0 was performed and analysed to obtain information on the interaction of ethanol with different parts of albumin molecule both fatty acid containing and fatty acid free. The differences in ethanol binding affinity for both kinds of albumin were found. At very low concentrations ethanol was observed to be a stabilizer of the folded state of albumin contrary to the higher concentration where its binding to the unfolded protein predominates.     

Bonding strength of fluorinated and hydrogenated surfactant to bovine serum albumin

The interactions between bovine serum albumin (BSA) and different surfactants are investigated by the fluorescence technique. Pairs of fluorinated and hydrogenated surfactants with similar hydrophobic chain lengths including potassium perfluorooctanesulfonate and sodium octanesulfonate are studied in order to determine their interactions with BSA. The binding constants and thermodynamic parameters between BSA and different surfactants are compared and the main binding strength is determined. The mechanism of quenching and change of particle size gives rise to the binding force. Based on the FRET theory, the distances between potassium perfluorooctanesulfonate/sodium octanesulfonate and BSA are calculated and it is found that the fluorinated surfactant exhibits stronger interactions with proteins than the hydrogenated one, which is also proved by zeta potential and TEM.     

Characterization of the mechanical properties of cross-linked serum albumin microcapsules: effect of size and protein concentration

A microfluidic technique is used to characterize the mechanical behavior of capsules that are produced in a two-step process: first, an emulsification step to form droplets, followed by a cross-linking step to encapsulate the droplets within a thin membrane composed of cross-linked proteins. The objective is to study the influence of the capsule size and protein concentration on the membrane mechanical properties. The microcapsules are fabricated by cross-linking of human serum albumin (HSA) with concentrations from 15 to 35 % (w/v). A wide range of capsule radii (～40–450 μm) is obtained by varying the stirring speed in the emulsification step. For each stirring speed, a low threshold value in protein concentration is found, below which no coherent capsules could be produced. The smaller the stirring speed, the lower the concentration can be. Increasing the concentration from the threshold value and considering capsules of a given size, we show that the surface shear modulus of the membrane increases with the concentration following a sigmoidal curve. The increase in mechanical resistance reveals a higher degree of cross-linking in the membrane. Varying the stirring speed, we find that the surface shear modulus strongly increases with the capsule radius: its increase is two orders of magnitude larger than the increase in size for the capsules under consideration. It demonstrates that the cross-linking reaction is a function of the emulsion size distribution and that capsules produced in batch through emulsification processes inherently have a distribution in mechanical resistance.     

Effect of bovine serum albumin on the surface properties of ionic liquid-type Gemini surfactant

The effect of bovine serum albumin on the surface properties of IL-type gemini surfactant ([C₁₀-4-C₁₀im]Br₂), have been investigated by surface tension method. The critical micelle concentration (CMC) as a function of BSA concentrations at various temperatures was investigated. The CMC of [C₁₀-4-C₁₀im]Br₂ increases with increasing the concentration of BSA as well as the temperature of the system. The interfacial parameters viz; maximum surface excess concentration (Γ_max), the minimum area per molecule (A_min), and surface pressure at CMC (Π_cmc) were calculated. In addition, thermodynamic parameters of adsorption and micellization were evaluated by using surface tension data. The results indicated that the binding of [C₁₀-4-C₁₀im]Br₂ to BSA is spontaneous and exothermic in nature. The process is entropy driven and hydrophobic interactions are the major driving forces.     

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C₄₄), 83% carbamylated (C₈₃) and 85% acetylated (A₈₅) were made by treating the HSA solution with a different molar excess of potassium cyanate and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl S-200 HR column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C₈₃ and A₈₅ HSAs while C₄₄ HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 ​nm to a different extent. Whereas the values of the Stern-Volmer constant (K_SV) and bimolecular quenching rate constant (k_q) suggested, NA-HSA complex formation, binding constant (K_a) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the K_a value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.     

Formation at low surfactant concentrations and characterization of mesoporous MCM-41

At low concentrations of cetyltrimethylammonium bromide,all silica-based mesoporous materials with hexagonal phase have been synthesized via interactions between self-assembled surfactant molecule aggregates and aniomc silicate polymers.The resulting materials are characterized by XRD,FT-IR,solid state 29Si MAS NMR,thermal analysis and N2 adsorption-desorption measurements.After soluble ions are removed,the interactors between surfactant micelles and silicate polymers are reorganized and then form mesostructures 1 he hexagonal framework is sonsistent with amorphous silica gel.The structures of materials depend on the synthesis conditions Hydrothermal process improves the interactions between molecules and increases the degree of framework silicon atom polymerization The.surface area and the mesopore volume of the material prepared at 100℃ increase by 87% and 71 %,respectively,compared with those obtained at room temperature.     

Solubilization of pristine fullerene by the unfolding mechanism of bovine serum albumin for cytotoxic application

A method for solubilization of pristine fullerene (C(60)) in water was proposed by directly using bovine serum albumin (BSA) as a "solubilizer" by means of the unfolding mechanism. C(60) aqueous solution with a small distribution size, excellent dispersion stability and high dispersion concentration was obtained without the use of derivatization and organic solvent.     

Spectroscopic study on the inherent binding information of cationic perfluorinated surfactant with bovine serum albumin

UV-vis, FT-IR, fluorescence and synchronous fluorescence spectra are applied to discuss the inherent binding information of model protein bovine serum albumin (BSA) with perfluorinated surfactant trimethyl-1-propanaminium iodide (FC-134). According to the results analyzed from Stern-Volmer equation, FC-134 can quench the fluorescence intensity of BSA via a dynamic quenching mechanism with complex formation. The thermodynamic parameters are calculated, revealing that hydrophobic force is the main interaction driven force. The binding constants and number of binding sites are also obtained. With the aid of site markers—warfarin and ibuprofen, we first report that FC-134 primarily binds to tryptophan residue Trp-214 of BSA within site I (sub-domain IIA).     

Study on the interaction between antibacterial drug and bovine serum albumin: A spectroscopic approach

The binding of sulfamethoxazole (SMZ) to bovine serum albumin (BSA) was investigated by spectroscopic methods viz., fluorescence, FT-IR and UV–vis absorption techniques. The binding parameters have been evaluated by fluorescence quenching method. The thermodynamic parameters, ΔH°, ΔS°and ΔG° were observed to be −58.0 kJ mol⁻¹, −111 J K⁻¹ mol⁻¹ and −24 kJ mol⁻¹, respectively. These indicated that the hydrogen bonding and weak van der Waals forces played a major role in the interaction. Based on the Forster's theory of non-radiation energy transfer, the binding average distance, r, between the donor (BSA) and acceptor (SMZ) was evaluated and found to be 4.12 nm. Spectral results showed the binding of SMZ to BSA induced conformational changes in BSA. The effect of common ions and some of the polymers used in drug delivery for control release was also tested on the binding of SMZ to BSA. The effect of common ions revealed that there is adverse effect on the binding of SMZ to BSA.     

Formation of water-in-CO(2) microemulsions with non-fluorous surfactant Ls-54 and solubilization of biomacromolecules

The solubility of Ls-54 surfactant in supercritical CO(2) was determined. It was found that the surfactant was highly soluble in SC CO(2) and the water-in-CO(2) microemulsions could be formed, despite it being a non-fluorous and non-siloxane nonionic surfactant. The main reasons for the high solubility and formation of the microemulsions may be that the surfactant has four CO(2)-philic groups (propylene oxide) and five hydrophilic groups (ethylene oxide) and its molecular weight are relatively low. The results of this work provide useful information for designing CO(2)-soluble non-fluorous and non-siloxane surfactants. The phase behavior of the CO(2)/Ls-54/H(2)O system, solvatochromic probe study, and the UV spectrum of lysozyme proved the existence of water domains in the SC CO(2) microemulsions. The method of synchrotron radiation small-angle X-ray scattering was used to obtain the structural information on the Ls-54 based water-in-CO(2) reverse micelles. By using the Guinier plot (ln I(q) versus q (2)) on the data sets in a defined small q range (0.022-0.040 A(-1)), the radii of the reverse micelles were obtained at different pressures and molar ratio of water to surfactant, W(0), which were in the range of 20.4-25.2 A.     

Peroxidatic activity of metalloporphyrin binding to serum albumin: Enhancement effect of serum albumin on metalloporphyrin catalyzed luminol chemiluminescence reaction

The metalloporphyrin (M-P) catalyzed luminol-H₂O₂ chemiluminescence (CL) system can be significantly enhanced in the presence of serum albumins. The enhancement may be explained in terms of the formation of a molecular complex between M-P and serum albumin. The hydrophobic and coordination interactions between M-P and serum albumin were confirmed by comparing the degree of enhancement by the protein on different types of M-P catalyzed CL reactions and by comparing the catalytic activity of M-P/bovine serum albumin (BSA) and M-P/BSA/anti-BSA complexes. Only the non-ionic M-P catalyzed CL reaction was significantly enhanced, and the enhanced CL reaction was inhibited by an immunoreaction. The optimum conditions of the M-P/albumin complex catalyzed CL reaction were evaluated by using a flow-injection system, which was very similar to that using hemoglobin as the catalyst. A brief prospective on the general applicability of the enhanced CL reaction for the determination of serum albumin (2.5–500 μg/ml) is given.     

A spectral approach to determine location and orientation of azo dyes within surfactant aggregates

The UV–vis absorption properties of azo dyes are known to exhibit a variation with the polarity and acidity of the dye environment. The spectral properties of a series of anionic azo dyes were characterized to further probe the interaction of these dyes with two types of surfactant aggregates: (1) the spherical micelles formed in aqueous solution by alkyltrimethylammonium bromide (C_nTAB) surfactants with n = 10–16 and (2) the unilamellar vesicles spontaneously formed in water from binary mixtures of the oppositely-charged double-tailed surfactants cationic didodecyldimethylammonium bromide (DDAB) and anionic sodium dioctylsulfosuccinate (Aerosol OT or AOT). The observed dye spectra reflect the solvatochromic behavior of the dyes and suggest the location and orientation of the dye within the surfactant aggregates. Deconvolution of the overall spectra into sums of Gaussian curves more readily displays any contributions of tautomeric forms of the azo dyes resulting from intramolecular hydrogen bonding. The rich variation in UV/vis absorption properties of these anionic azo dyes supports their use as sensitive tools to explore the nanostructures of surfactant aggregates.     

Bovine serum albumin (BSA) plays a role in the size of SDS micelle-like aggregates at the saturation binding: the ionic strength effect

The influence of ionic strength on the complexes formed by natural bovine serum albumin (BSA), pH 5.4 (near the isoelectric point), and sodium dodecyl sulfate (SDS) in aqueous buffered (sodium acetate) solution was investigated by using surface tension, fluorescence and small angle X-ray scattering (SAXS) techniques. Ionic strength was varied by changing sodium acetate buffer concentration from 0.020 to 0.5 M. Surface tension revealed that SDS:BSA saturation binding occurs at psp = 42 +/- 2 mM, independent of the solution ionic strength. Further, SAXS curves are consistent with the necklace and bead model, where micelle-like aggregates are randomly distributed along the partial unfolded protein. Micelle-like aggregates grow from small spheres at 10 mM SDS to small ellipsoids (upsilon = 1.3 , ratio between the largest and the shortest axes) near psp, in good agreement with micellar aggregation numbers obtained by fluorescence, independent of salt concentration. Protein-bound micelles stop growing above psp and further SDS addition induces free-micelle formation.     

Polyoxometalate binding to human serum albumin: a thermodynamic and spectroscopic approach

The molecular recognition of polyoxometalates by human serum albumin is studied using two different polyoxometalates (POMs) at pH 7.5. The results are compared with those obtained at pH 3.5 and 9.0. At pH 7.5, both POMs strongly interact with the protein with different binding behaviors. The Keggin shaped POM, [H(2)W(12)O(40)](6-) (H2W12), specifically binds the protein, forming a complex with a 1:1 stoichiometry with Ka = 2.9 x 10(6) M(-1). The binding constant decreased dramatically with the increase of the ionic strength, thus indicating a mostly electrostatic binding process. Isothermal titration calorimetry (ITC) experiments show that the binding is an enthalpically driven exothermic process. For the wheel shaped POM [NaP(5)W(30)O(110)](14-) (P5W30), there are up to five binding sites on the protein. Increasing the ionic strength changes the binding behavior significantly, leading to a simple exothermic process, with several binding sites. Competitive binding experiments indicate that the two POMs share one common binding site. In addition, they show the existence of another important binding site for P5W30. The two POMs exhibit different binding dependences on the pH. The combination of the experimental results with the knowledge of the surface map of the protein in its N-B conformation transition domain leads to the proposal for the probable binding site of POMs. The present work reveals a protein conformation change upon P5W30 binding, a new feature not explicitly documented in previous studies.