Exploring the Conformation and Thermal Stability of Human Serum Albumin Corona of Ferrihydrite Nanoparticles.

In the last few years, a great amount of attention has been given to nanoparticles research due to their physicochemical properties that allow their use in analytical instruments or in promising imaging applications on biological systems. The use of ferrihydrite nanoparticles (Fh-NPs) in practical applications implies a particular control of their magnetic properties, stability, biocompatibility, interaction with the surface of the target, and low toxicity. In this study, the formation and organization of human serum albumin (HSA) molecules around the simple Fh-NPs and Fh-NPs doped with Co and Cu were examined by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) in terms of morphology and particle size. The topology of all Fh-NPs shows an organized area of HSA around each type of Fh-NP. Molecular docking studies were used in order to determine the probable location of the ferrihydrite in the HSA structure. The thermal stability of these nanohybrids was further investigated by fluorimetry, using 214-Trp residue from HSA as a spectral sensor. The denaturation temperature (Tm) was determined, and stabilization of the HSA structure in the presence of Fh-NPs was discussed. This study could be a starting point for the development of different applications targeting the structure and stability of Fh-NPs complexes with proteins.

Antiamyloidogenic Effects of Ellagic Acid on Human Serum Albumin Fibril Formation Induced by Potassium Sorbate and Glucose.

Oxidative stress has the main role in protein conformational changes and consequent direct involvement in different kind of diseases. Potassium sorbate as a widespread industrial preservative and glucose are two important oxidants that can be involved in oxidative stress. In this study the effect of ellagic acid as a phenolic antioxidant on amyloid fibril formation of human serum albumin upon incubation of potassium sorbate and glucose was studied using thioflavin T assay, surface tension, atomic force microscopy, Amadori product, and carbonyl content assays. The thioflavin T assay and atomic force microscopy micrographs demonstrated the antiamyloidogenic effect of ellagic acid on the human serum albumin fibril formation. This antioxidant also had the repair effect on surface tension of the modified human serum albumin (amyloid intermediates), which was destructed, caused by potassium sorbate and glucose. This mechanism takes place because of potent carbonyl stress suppression effect of ellagic acid, which was strengthening by potassium sorbate in the presence and absence of glucose.

All-trans-retinoic acid and retinol binding to the FA1 site of human serum albumin competitively inhibits heme-Fe(III) association.

Retinoids are a class of chemicals derived from vitamin A metabolism, playing important and diverse functions. Vitamin A, also named all-trans-retinol (all-trans-ROL), is coverted into two classes of biologically active retinoids, i.e. 11-cis-retinoids and acidic retinoids. Among acidic retinoids, all-trans-retinoic acid (all-trans-RA) and 9-cis-retinoic acid (9-cis-RA) represent the main metabolic products. Specific and aspecific proteins solubilize, protect, and detoxify retinoids in the extracellular environment. The retinoid binding protein 4 (RBP4), the epididymal retinoid-binding protein (ERBP), and the interphotoreceptor matrix retinoid-binding protein (IRBP) play a central role in ROL transport, whereas lipocalin-type prostaglandin D synthase (also named ß-trace) and human serum albumin (HSA) transport preferentially all-trans-RA. Here, the modulatory effect of all-trans-RA and all-trans-ROL on ferric heme (heme-Fe(III)) binding to HSA is reported. All-trans-RA and all-trans-ROL binding to the FA1 site of HSA competitively inhibit heme-Fe(III) association. Docking simulations and local structural comparison of HSA with all-trans-RA- and all-trans-ROL-binding proteins support functional data indicating the preferential binding of all-trans-RA and all-trans-ROL to the FA1 site of HSA. Present results may be relevant in vivo, in fact HSA could act as a secondary carrier of retinoids in human diseases associated with reduced levels of RBP4 and IRBP.

Spectroscopic Studies on the Molecular Ageing of Serum Albumin.

Pathological states in the organism, e.g., renal or hepatic diseases, cataract, dysfunction of coronary artery, diabetes mellitus, and also intensive workout, induce the structural modification of proteins called molecular ageing or N-A isomerization. The aim of this study was to analyze the structural changes of serum albumin caused by alkaline ageing using absorption, spectrofluorescence, and circular dichroism spectroscopy. The N-A isomerization generates significant changes in bovine (BSA) and human (HSA) serum albumin subdomains-the greatest changes were observed close to the tryptophanyl (Trp) and tyrosyl (Tyr) residue regions while a smaller change was observed in phenyloalanine (Phe) environment. Moreover, the changes in the polarity of the Trp neighborhood as well as the impact of the ageing process on α-helix, ß-sheet content, and albumin molecule rotation degree have been analyzed. Based on the spectrofluorescence study, the alterations in metoprolol binding affinity to the specific sites that increase the toxicity of the drug were investigated.

Exploring the copper(II)-aminotriazole complex-binding sites of human serum albumin.

The potential impact on human exposure to aminotriazole (ATA) and heavy metal in the environment becomes a concerning issue. In the current study, a water-soluble Cu(II)-aminotriazole complex [Cu(II)-ATA] was synthesized. To explore the binding mechanism of the complex with human serum albumin (HSA), their effects on conformation and activity of HSA by multispectroscopic approach and molecular modeling were investigated. Further fluorescent tests revealed that the quenching mechanism of HSA by Cu(II)-ATA was overall static. Meanwhile, the obtained binding constant and thermodynamic parameters on complex-HSA interaction showed that the types of interaction force of Cu(II)-ATA and HSA were hydrogen bonding, van der Waals and electrostatic. The analysis of three-dimensional fluorescence, circular dichroism and Fourier transform infrared spectroscopy showed that Cu(II)-ATA induced the changes in the secondary structure of HSA. Molecular docking simulation was performed and docking model suggested that the complex docked into HSA at subdomain IIA. Furthermore, amino group and attractive electrostatic interaction of Cu(II)-ATA greatly contributed to the hydrogen bonding, van der Waals and electrostatic interaction between Cu(II)-ATA and HSA, as confirmed by experimental data.

Fatty acid binding into the highest affinity site of human serum albumin observed in molecular dynamics simulation.

Multiple molecular dynamics simulations were performed to investigate the association of stearic acid into the highest affinity binding site of human serum albumin. All binding events ended with a rapid (<10 ps) lock-in of the fatty acid due to formation of a hydrogen bond with Tyr401. The kinetics and energetics of the penetration process both depended linearly on the positional shift of the fatty acid, with an average insertion time and free energy reduction of, respectively, 32 ± 20 ps and 0.70 ± 0.15 kcal/mol per methylene group absorbed. Binding events of longer duration (tbind>1 ns) were characterized by a slow exploration of the pocket entry and, frequently, of a nearby protein crevice corresponding to a metastable state along the route to the binding site. Taken all together, these findings reconstruct the following pathway for the binding process of stearic acid: (i) contact with the protein surface, possibly facilitated by the presence of an intermediate location, (ii) probing of the site entry, (iii) insertion into the protein, and (iv) lock-in at the final position. This general description may also apply to other long-chain fatty acids binding into any of the high-affinity sites of albumin, or to specific sites of other lipid-binding proteins.

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.

Influence of MLS laser radiation on erythrocyte membrane fluidity and secondary structure of human serum albumin.

The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm(-2) and surface energy power density 195 mW cm(-2) (1,000 Hz) and 230 mW cm(-2) (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.

Stretched extracellular matrix proteins turn fouling and are functionally rescued by the chaperones albumin and casein.

While evidence is mounting that cells exploit protein unfolding for mechanochemical signal conversion (mechanotransduction), what mechanisms are in place to deal with the unwanted consequences of exposing hydrophobic residues upon force-induced protein unfolding? Here, we show that mechanical chaperones exist that can transiently bind to hydrophobic residues that are freshly exposed by mechanical force. The stretch-upregulated binding of albumin or casein to fibronectin fibers is reversible and does not inhibit fiber contraction once the tension is released.

Detection of Pulmonary Embolism with Gallium-68 Macroaggregated Albumin Perfusion PET/CT: An Experimental Study in Rabbits.

This study was designed to evaluate the accuracy of detecting pulmonary embolism (PE) using the Technegas SPECT/CT combined with 68Ga PET/CT in a rabbit model. One hour after artificial PE (n = 6) and sham (n = 6) models were created, Technegas SPECT/CT ventilation and 68Ga-MAA PET/CT perfusion scan (V/Q scan) were performed. Ventilation imaging was performed first on all cases. Technegas SPECT/CT and 68Ga-MAA PET/CT images were evaluated by a nuclear medicine physician who recorded the presence, number, and location of PE on a per-lobe basis. The sensitivity, specificity, and accuracy of Technegas SPECT/CT and 68Ga-MAA PET/CT for detecting PE were calculated using a histopathological evaluation as a reference standard. A total of 60 lung lobes were evaluated in 12 rabbits, and PE was detected in 20 lobes in V/Q scans and histopathological analysis. The overall sensitivity, specificity, and accuracy were 100%, 100%, and 100%, respectively, for both the Technegas SPECT/CT and 68Ga-MAA PET/CT V/Q scans. Technegas/68Ga-MAA V/Q scans have good sensitivity, specificity, and accuracy in the detection of PE in this animal model study.

Dynamic analysis of the atomic vibrations of proteins, as exemplified by the binding of myristic acid to human serum albumin.

Prediction of the biological function of a protein from its three-dimensional structure is an important, still unsolved problem. A new approach to this objective, tried here, is use of crystallographic temperature factors, which contain the same information as IR and Raman spectra but lack their overlap problems. The hypothesis that atomic vibrations are evolutionally optimized for a particular function by adoption of collective modes governed by an attractor has been tested on 19 proteins with the result that strong correlation (r = 0.98) was found between the dimension of the attractor and the number of vectors needed to describe the function. The binding of five molecules of myristic acid (MA) to human serum albumin (HSA) at two sites accommodating two or three MA molecules, respectively, gave rise to four conformational changes in distinct regions. Two of these were located at the binding sites but the others occurred in segments far removed from the ligands both in the sequence and spatially. According to the statistical criteria employed, the conformational changes at the ligand-binding sites were not necessarily controlled by an attractor of low order, but the others were governed by one of dimension of 2-3. This was ascribed to entropic compensation. The results were tested using another ligand, an inhibitor of the BCL-2 family of proteins. The HSA underwent the same conformational changes with this ligand as with MA.

Protein fluorescence decay: a gamma function description of thermally induced interconversion of amino acid rotamers.

We present a description of fluorescence decay kinetics in complex environments based on gamma functions rather than the conventional approach using exponentials. The gamma function description is tested in measurements on the temperature dependence of the protein human serum albumin (HSA), N-acetyl tryptophanamide (NATA), and 2, 5-dipenyl oxazole (PPO). The monitoring of macromolecular structure and dynamics is demonstrated by means of distinct tryptophan (Trp) rotamer populations and their interconversion in HSA.

Potential protein toxicity of synthetic pigments: binding of poncean S to human serum albumin.

Using various methods, e.g., spectrophotometry, circular dichroism, and isothermal titration calorimetry, the interaction of poncean S (PS) with human serum albumin (HSA) was characterized at pH 1.81, 3.56, and 7.40 using the spectral correction technique, and Langmuir and Temkin isothermal models. The consistency among results concerning, e.g., binding number, binding energy, and type of binding, showed that ion pair electrostatic attraction fixed the position of PS in HSA and subsequently induced a combination of multiple noncovalent bonds such as H-bonds, hydrophobic interactions, and van der Waals forces. Ion pair attraction and H-bonds produced a stable PS-HSA complex and led to a marked change in the secondary structure of HSA in acidic media. The PS-HSA binding pattern and the process of change in HSA conformation were also investigated. The potentially toxic effect of PS on the transport function of HSA in a normal physiological environment was analyzed. This work provides a useful experimental strategy for studying the interaction of organic substances with biomacromolecules, helping us to understand the activity or mechanism of toxicity of an organic compound.

Imprinting effect of protein-imprinted polymers composed of chitosan and polyacrylamide: a re-examination.

In this paper the imprinting effects recently reported with protein-imprinted polymers based on chitosan and polyacrylamide are re-assessed. Molecularly imprinted polymers (MIPs) from which the embedded template molecules were removed by washing with a solution of sodium dodecyl sulfate and acetic acid were prepared. In batch template rebinding experiments, the MIPs displayed quite high template binding capacity as reported previously. However, the non-imprinted polymers (NIPs), after washing with the same solution, also showed large binding capacity nearly equal to that of the MIPs. X-ray diffraction and scanning electronic microscope investigations confirmed remarkable property changes of the NIPs after the washing process. These findings indicate that the non-specific adsorption resulting from the template removing process rather than the imprinted sites generated on the MIPs themselves may account for the high template rebinding capacity of the reported protein-imprinted polymers.

Energetics of the interactions of human serum albumin with cationic surfactant.

The heat capacity changes for interaction of human serum albumin (HSA) and a cationic surfactant-cetylpyridinium chloride (CPC), were studied at conditions close to physiological (50mM HEPES or phosphate buffer, pH 7.4 and 160mM NaCl) carrying out isothermal calorimetric titrations (ITC) at various temperatures (20-40 degrees C). ITC measurements indicated that the small endothermic changes associated with CPC demicellization were temperature independent at these conditions. Surprisingly, important enthalpy changes associated with binding of CPC to HSA were exothermic and temperature independent at lower concentrations (below 0.022mM) of CPC and endothermic and temperature dependent at higher concentrations of CPC. The values of heat capacity changes were obtained for each studied concentration of CPC from the plot of enthalpy changes vs temperature. The obtained results demonstrate the temperature independence of heat capacity changes at entire range of studied CPC concentrations. Both enthalpograms and heat capacity curves indicate the two-step mechanism of HSA folding changes due to its interactions with CPC. The first step corresponds to transition from native state to partially unfolded state and the second to unfolding and to the loss of tertiary structure. The analysis of the results indicates that predominant cooperative unfolding occurs at CPC/HSA molar ratio region between 25 and 30. Such information could not be extracted from thermograms and describes the role of heat capacity as a major thermodynamic quantity giving insight on physical, mechanistic and even atomic-level into how HSA may unfold and interact with CPC. The effect of CPC binding on HSA intrinsic fluorescence, UV-Vis and CD spectra were also examined. Hence, the analysis of spectral data confirms the ITC results about the biphasic mechanism of HSA folding changes induced by CPC. The CD measurement also represents the conservation of considerable secondary structure of HSA due to interaction with CPC.

Biophysical insight into the interaction mechanism of plant derived polyphenolic compound tannic acid with homologous mammalian serum albumins.

Numerous phenolic compounds have been reported in the last decade that have a good antioxidant property and interaction affinity towards mammalian serum albumins. In the present study, we have utilized mammalian serum albumins as a model protein to examine their comparative interaction property with polyphenolic compound tannic acid (TA) by using various spectroscopic and calorimetric methods We have also monitored the esterase and antioxidant activity of mammalian serum albumins in the absence and presence of TA. The obtain results recommended that the TA have a good binding affinity (∼104 to 106M-1) towards mammalian serum albumins and shows double sequential binding sites, which depends on the concentration of TA that induced the conformational alteration which responsible for the thermal stability of proteins. Binding affinity, structural transition and thermodynamic parameters were calculated from spectroscopic and calorimetric method reveals that non-covalent interaction causes partial conformational alteration in the secondary structure of protein ie.; increase in α-helical content with decrease in ß-sheet, random coil and other structure. Meanwhile, we have found that esterase activities of serum albumins were also stabilized against hydrolysis and shows higher antioxidant activity in the presence of TA because albumins its self have an immense antioxidant activity beside TA.

An Alternatively Packed Dry Molten Globule-like Intermediate in the Native State Ensemble of a Multidomain Protein.

It has been difficult to quantify the degree of side-chain conformational heterogeneity in the native (N) state ensemble of proteins and the relative energetic contributions of the side-chain packing and the hydrophobic effect in protein stability. Here, we show using multiple site-specific spectroscopic probes and tools of thermodynamics that the N state ensemble of a multidomain protein contains an equilibrium intermediate (I) whose interdomain region resembles a dry molten globule. In the I state, a tryptophan residue in the interdomain region is alternatively packed, but its secondary structure and intradomain packing are N-like. The I state also has a larger interdomain distance, but the domain-domain interface is dry and molten. Our results indicate that hydrophobic desolvation and side-chain packing are decoupled during protein folding and that interdomain packing interactions have an important energetic contribution in protein stability. Dynamic interconversion between alternatively packed N-like states could be important for multiple allosteric and ligand binding functions of this protein.

A simple improved desolvation method for the rapid preparation of albumin nanoparticles.

The current study tried to establish a simple and fast method for the preparation of BSA and HSA nanoparticles, based on an improved desolvation procedure under the aspect of a controllable particle size around 100nm for drug delivery applications. The Procedure used for the nanoparticles preparation was simplified by using a designed apparatus for controlling the addition of ethanol and it was used instead of conventional tubing pump which enabled the preparation of nanoparticles under defined conditions. By using EDC as cross-linker instead of glutharaldehyde, the time of nanoparticles preparation procedure was reduced to 3h. Several factors of the preparation process, such as the volume of the albumin solution, desolvating agent volume, the amount of cross-linker, the presence of salts and protein concentration were evaluated. Nanoparticles with smaller size were obtained under experimental conditions without the presence of salts or the use of buffers, 250mg of protein/4ml water, 5mg cross-linker, the addition of 4 and 8ml ethanol by using the designed apparatus to the HSA and BSA solution, respectively. By using this improved method, BSA and HSA nanoparticles of the size around 100nm and polydispersity below 0.2 were obtained.

Coating alginate microspheres with a serum albumin-alginate membrane: application to the encapsulation of a peptide.

Calcium alginate gel microspheres coated with a human serum albumin (HSA)-alginate membrane were prepared adapting a transacylation method previously applied to large beads. The procedure involved emulsification of an aqueous solution of sodium alginate and propylene glycol alginate (PGA) in an oily phase, followed by addition of CaCl(2). The resulting gel microspheres were transferred in an aqueous solution of HSA. The addition of 0.5 M NaOH started the reaction between PGA and HSA, producing amide bonds and forming a membrane around the particles. An optimization study was conducted, notably exploring the addition of HSA to the internal phase. The microcapsules were studied with respect to morphology (optical and scanning electron microscopy) and size (laser granulometry), in comparison with uncoated gel microspheres. Biocompatibility was checked in osteoblast cultures. Lysine-arginine-phenylalanine-lysine (KRFK) was encapsulated and the release kinetics was studied in vitro. The method provided stable microspheres (size around 60 microm), with a membrane surviving a treatment with citrate and resisting lyophilization. The microcapsules were shown biocompatible. The release of KRFK was slower (release time>8 days) than that of uncoated microspheres. These microcapsules might be useful as peptide containers to be combined with prosthetic materials for improving osteointegration.

Effect of glass dissolution products on the detection of proteins by silver staining.

The influence of glass dissolution on the silver staining of proteins was investigated by reacting glass microspheres of varying chemical durability in boiling Laemmli sample buffer (LSB) for up to 5 min. All three of the investigated glass compositions leached Na+ ions to varying degrees during boiling in LSB, thereby causing an increase in the pH of the sample buffer. The LSB supernatant from the dissolution tests was mixed with unreacted LSB containing human serum albumin (HSA) and standard one-dimensional SDS-PAGE was performed. Silver staining was then used to visualize protein bands within the gel. The 30 Na2O.70 SiO2 glass exhibited pronounced degradation as shown by scanning electron microscopy. Further experiments employing solutions of neat LSB and reacted LSB (i.e., LSB containing glass dissolution products) mixed at varying ratios demonstrated the apparent significance of sample pH in affecting the inhibition of silver staining. The cause of this behavior may be due to an interference with the fixation stage of the staining protocol, thereby resulting in the loss of protein in subsequent rinsing stages.