Structure Of BSA Research Articles

Understanding the structural parameters of biocompatible nanoparticles dictating protein fouling.

The development of nanocarriers for biomedical applications requires that these nanocarriers have special properties, including resistance to nonspecific protein adsorption. In this study, the fouling properties of PLA- and PCL-based block copolymer nanoparticles (NPs) have been evaluated by placing them in contact with model proteins. Block copolymer NPs were produced through the self-assembly of PEOm-b-PLAn and PEOm-b-PCLn. This procedure yielded nanosized objects with distinct structural features dependent on the length of the hydrophobic and hydrophilic blocks and the volume ratio. The protein adsorption events were examined in relation to size, chain length, surface curvature, and hydrophilic chain density. Fouling by BSA and lysozyme was considerably reduced as the length of the hydrophilic PEO-stabilizing shell increases. In contrast to the case of hydrophilic polymer-grafted planar surfaces, the current investigations suggest that the hydrophilic chain density did not markedly influence protein fouling. The protein adsorption took place at the outer surface of the NPs since neither BSA nor lysozyme was able to diffuse within the hydrophilic layer due to geometric restrictions. Protein binding is an exothermic process, and it is modulated mainly by polymer features. The secondary structures of BSA and lysozyme were not affected by the adhesion phenomena.

A rare dihydroxo copper(ii) complex with ciprofloxacin; a combined experimental and ONIOM computational study of the interaction of the complex with DNA and BSA

A rare mononuclear Cu(II) complex, trans-[Cu(cip)2(OH)2]·2CH3OH·6H2O, where cip+/− is a zwitterionic form (+H2N–⋯–COO−) of ciprofloxacin, has been synthesized and characterized by spectroscopic methods and X-ray crystallography. In vitro studies (UV-Vis spectroscopy, emission titration, voltammetric techniques, and gel electrophoresis) show that the complex interacts with calf-thymus DNA (CT-DNA) via an intercalative binding mode. The microenvironment and the secondary structure of BSA are changed in the presence of the complex. Also, our Own N-layered Integrated molecular Orbital and molecular Mechanics (ONIOM) method, based on the hybridization of QM/MM methodology, was carried out to investigate the interaction of the complex with DNA and BSA. The calculated electronic interaction energy of the complex with DNA in the gas phase and water is −64.358 and −56.685 kcal mol−1, respectively. The calculated interaction of the complex with BSA is −54.231 kcal mol−1. The theoretical data confirm the experimental results with respect to the mechanism of binding and binding constants. Finally, the calculations indicate that the QM/MM is a more precise method than the docking method for determination of the electronic interaction energies because of two reasons: (I) it considers the complex quantum mechanically in calculations, and (II) it accounts for the polarization of the wave function of the complex by the charge distribution of the molecular mechanical part (DNA and BSA).

Binding of (−)-epigallocatechin-3-gallate with thermally-induced bovine serum albumin/ι-carrageenan particles

Novel thermally-induced BSA/ι-carrageenan particles are used as a protective carrier for (−)-epigallocatechin-3-gallate (EGCG). The addition of EGCG to BSA/ι-carrageenan particles can highly quench the intrinsic fluorescence of BSA, which is explained in terms of the binding of EGCG to the hydrophobic pockets of BSA mainly through the hydrophobic force. According to the double logarithm equation, the binding constant is determined as 1.1×108M−1 for the binding of EGCG with BSA/ι-carrageenan particles. The high binding affinity is ascribed to both the molecular structure of EGCG and the partial unfolding state of BSA in BSA/ι-carrageenan particles. The circular dichroism spectra and calculated α-helix of BSA suggest that the bound EGCG leads to a more random secondary structure of BSA. Furthermore, BSA/ι-carrageenan particles are found to be superior to native BSA and pure BSA particles for improving the stability and radical scavenging activity of EGCG.

Exploring the role of ligand-BSA in the response of BSA-protected gold-nanoclusters to silver (I) Ions by FT-IR and circular dichroism spectra

Previously, we have explored the mechanism of the response of BSA-protected small gold nanoclusters (Au16NCs@BSA) to silver (I) ions (Ag+) by using XPS, but the role of the ligand BSA in this response was not clear. Therefore, we used FT-IR and circular dichroism (CD) spectra to monitor the changes of the secondary structure of ligand BSA. After adding Ag+ to the AuNCs@BSA, compare with the native BSA, the ligand-BSA showed little differences in the position of main peaks but more differences in the profile of this peak in FT-IR spectra. While in CD spectra it is not only peak shape changed but also peak position. All the results showed silver ions can bind to ligand BSA, and induced their secondary structure changes. But the changes of ligand BSA are not enough to influence the fluorescence emission of AuNCs@BSA, especially for the emission of AuNCs. And BSA-protected different size gold nanoclusters have the similar changes in spatial structure of ligand BSA, but only the Au16NCs@BSA could response to Ag+, which indicated that the ligand BSA was not the key role for the special fluorescent response.

In vitro cytotoxicity studies of palladacyclic complexes containing the symmetric diphosphine bridging ligand. Studies of their interactions with DNA and BSA

The reactions between [Pd2{(C,N)–C6H4CH2NH(Et)}2(μ-X)2] (X = Cl or Br) and 1,2-bis(diphenylphosphino)ethane (dppe) in the 1:1 molar ratio resulted in the dppe-bridged Pd(II) complexes, [Pd2{(C,N)–C6H4CH2NH(Et)}2(μ-dppe)(Cl)2] (1) and [Pd2{(C,N)–C6H4CH2NH(Et)}2(μ-dppe)(Br)2] (2), respectively, which were characterized by elemental analyses, infrared (IR), 1H- and 31P{1H} NMR spectroscopy. The molecular structure of 1 was determined by single-crystal X-ray diffraction. In vitro cytotoxicity of 1, 2, dppe, PhCH2NH(Et) and cisplatin were carried out against four human tumor cell lines. The interactions of complexes towards DNA and protein are investigated. The results suggested that both complexes could interact with FS-DNA through the intercalation mode. Moreover, the reactivity towards BSA revealed that the microenvironment and the secondary structure of BSA were changed in the presence of Pd(II) complexes.

Experimental and molecular modeling studies on the interaction of the Ru(II)-piroxicam with DNA and BSA

A mononuclear Ru(II) complex containing two piroxicam (Pir−) ligands was synthesized and fully characterized. Interaction studies of the Pir− anion and the Ru(II) complex with DNA and BSA were carried out using spectroscopic techniques. The results suggested that the Pir− anion binds to DNA in a moderately strong fashion via intercalation between the base stacks of double–stranded DNA, while the Ru(II) complex is a groove binder and interacts with DNA with more affinity. Moreover, the results demonstrated that the microenvironment and the secondary structure of BSA were changed in the presence of Pir‾ and Ru(II) complex. The free Pir‾ ligand and the Ru(II) complex can lead to the photocleavage of DNA supercoiled pUC57. Finally, the binding of the Ru(II) complex to BSA and DNA was modeled by molecular docking and molecular dynamic simulation methods.

Covalent Albumin Microparticles as an Adjuvant for Production of Mucosal Vaccines against Hepatitis B

Covalently modified albumin (BSA) microparticles were developed for potential use as an adjuvant in mucosal vaccines against hepatitis B. To synthesize consistent protein particles, a covalent approach was proposed to modify BSA. Our strategy was to bond maleic anhydride (MA) molecules to BSA structure by nucleophilic reaction for further radical cross-linking/polymerization reaction with N',N'-dimethylacrylamide (DMAAm). The presence of poly(N',N'-dimethylacrylamide) in the protein network enables the microparticles to show well-defined, homogeneous forms. Cytotoxicity tests showed that the cytotoxic concentration for 50% of VERO cells (CC50) was 216.25 ± 5.30 μg mL(-1) in 72 h of incubation. The obtained CC50 value is relatively low for an incubation time of 72 h, suggesting an acceptable biocompatibility. Assay of total protein showed that the encapsulation efficiency of the microparticles with hepatitis B surface antigen (HBsAg) was 77.7 ± 0.2%. For the reference sample, which was incubated without HBsAg, the quantity of protein was below the limit of detection.

Binding of Curcumin with Bovine Serum Albumin in the Presence of ι-Carrageenan and Implications on the Stability and Antioxidant Activity of Curcumin

This work studied the influences of formation of BSA/ι-carrageenan complexes on the binding, stability, and antioxidant activity of curcumin. In the presence of BSA and ι-carrageenan, curcumin gives higher intensities of absorption and fluorescence than free curcumin and curcumin only combined with BSA. The added ι-carrageenan is observed to promote curcumin for quenching the instrinsic fluorescence of BSA. These results are explained in terms of the formation of BSA/ι-carrageenan complexes, which help to stabilize the folded structure of BSA for providing curcumin with a more hydrophobic microenvironment. The small difference in anisotropy values of curcumin with BSA alone and of BSA/ι-carrageenan complexes suggests that ι-carrageenan acts as outer stretch conformation in BSA/ι-carrageenan complexes but does not directly disturb the hydrophobic pockets inside BSA, where curcumin is hydrophobically located. The determined values of the binding constant are higher for curcumin with BSA/ι-carrageenan complexes than with BSA alone. Moreover, BSA/ι-carrageenan complexes are found to be superior to single BSA for enhancing the stability and DPPH radical-scavenging ability of curcumin.

Ternary copper(ii)-polypyridyl enantiomers: aldol-type condensation, characterization, DNA-binding recognition, BSA-binding and anticancer property

Chiral enantiomers [Cu(phen)(L-threo)(H2O)]NO3 1 and [Cu(phen)(D-threo)(H2O)]NO3 2 (threo = threoninate) underwent aldol-type condensation with formaldehyde, with retention of chirality, to yield their respective enantiomeric ternary copper(II) complexes, viz. L- and D-[Cu(phen)(5MeOCA)(H2O)]NO3·xH2O (3 and 4; phen = 1,10-phenanthroline; 5MeOCA = 5-methyloxazolidine-4-carboxylate; x = 0-3) respectively. These chiral complexes were characterized by FTIR, elemental analysis, circular dichroism, UV-Visible spectroscopy, fluorescence spectroscopy (FL), molar conductivity measurement, ESI-MS and X-ray crystallography. Analysis of restriction enzyme inhibition by these four complexes revealed modulation of DNA binding selectivity by the type of ligand, ligand modification and chirality. Their interaction with bovine serum albumin was investigated by FL and electronic spectroscopy. With the aid of the crystal structure of BSA, spectroscopic evidence suggested their binding at the cavity containing Trp134 with numerous Tyr residues in subdomain IA. The products were more antiproliferative than cisplatin against cancer cell lines HK-1, MCF-7, HCT116, HSC-2 and C666-1 except HL-60, and were selective towards nasopharyngeal cancer HK-1 cells over normal NP69 cells of the same organ type.

Effect of protein thermo aggregation on the binding of BSA to gelatin type A

We study the effect of limited heat-induced aggregation of BSA on structure development in the water–gelatin-thermally aggregated BSA (BSATA) system. The pH is set at 5.4 and the temperature is higher than the conformation transition temperature of gelatin, but lower than the denaturation temperature of BSA. Dynamic light scattering, circular dichroism, and fluorescence measurements are used to monitor structure changes. Interaction of gelatin with BSATA leads to formation of large complex particles with an average radius ∼1500nm. BSA–gelatin complex formation accompanies partial destabilization of the secondary and tertiary structures of BSA and an additional exposure of hydrophobic tryptophan residues on the surface of the globule. It is shown that electrostatic interaction of the oppositely charged groups of BSATA and gelatin is responsible for formation of such complex particles, whereas the secondary forces (hydrophobic interaction and hydrogen bonds) play an important role in stabilization of the complex particles. The zeta potentials of the native and the thermally aggregated BSA samples were determined, and the solvent quality has been quantified by determining the activity of the protein samples in their saturated solutions. It was shown that steric reasons (large size of the thermally aggregated BSATA particles), and uncomplete charge compensation of the positively charged gelatin molecules by the negatively charged BSATA particles are the main factors in determining structure formation, while the levels of the activity of the native BSA and BSATA have a smaller effect on the structure of complex.

Chitosan Microneedle Patches for Sustained Transdermal Delivery of Macromolecules

This paper introduces a chitosan microneedle patch for efficient and sustained transdermal delivery of hydrophilic macromolecules. Chitosan microneedles have sufficient mechanical strength to be inserted in vitro into porcine skin at approximately 250 μm in depth and in vivo into rat skin at approximately 200 μm in depth. Bovine serum albumin (BSA, MW=66.5 kDa) was used as a model protein to explore the potential use of chitosan microneedles as a transdermal delivery device for protein drugs. In vitro drug release showed that chitosan microneedles can provide a sustained release of BSA for at least 8 days (approximately 95% of drugs released in 8 days). When the Alexa Fluor 488-labeled BSA (Alexa 488-BSA)-loaded microneedles were applied to the rat skin in vivo, confocal microscopic images showed that BSA can gradually diffuse from the puncture sites to the dermal layer and the fluorescence of Alexa 488-BSA can be observed at the depth of 300 μm. In addition, encapsulation of BSA within the microneedle matrix did not alter the secondary structure of BSA, indicating that the gentle nature of the fabrication process allowed for encapsulation of fragile biomolecules. These results suggested that the developed chitosan microneedles may serve as a promising device for transdermal delivery of macromolecules in a sustained manner.

A facile and green ultrasonic-assisted synthesis of BSA conjugated silver nanoparticles

The formation and growth of hybrid nanoparticles of a protein BSA and silver by ultrasonic assistance were tracked by surface plasmon resonance signal of silver nanoparticles and light scattering. The hybrid nanoparticles were characterized by surface plasmon resonance spectra, light scattering, TEM, circular dichroism spectroscopy and zeta potential. Size along with the spherical shape of the nanoparticles could be controlled and nanoparticles with diameters ranging from 8 to 140nm could be obtained, depending upon the ultrasonication time (15–30min) and molar ratio of AgNO3/BSA (20–200). The role of single free thiol group in the reduction of silver ions was also investigated by using DTNB modified BSA and protein conjugated silver nanoparticles were formed even with thiol modified BSA. The growth and size of the nanoparticles were governed by ultrasonic assisted Ostwald ripening. BSA conjugated with silver nanoparticles showed changes in the secondary structure with an increase in the beta sheet structure to 33% as compared to 7% in native BSA as determined by CD spectra. Zeta potential measurements in the pH range of 2.0–12.0 demonstrated that the surface charges of the BSA conjugated silver nanoparticles were similar to that of native BSA suggesting that surface charges and overall three dimensional structure of BSA did not change much. This approach provides a strategy for completely green synthesis of hybrid nanoparticles consisting of a biological entity and an inorganic material. This is the first application of ultrasonic assistance in formation of such hybrid nanomaterials in aqueous media.

Can electromagnetic fields influence the structure and enzymatic digest of proteins? A critical evaluation of microwave-assisted proteomics protocols

This study reevaluates the putative advantages of microwave-assisted tryptic digests compared to conventionally heated protocols performed at the same temperature. An initial investigation of enzyme stability in a temperature range of 37–80 °C demonstrated that trypsin activity declines sharply at temperatures above 60 °C, regardless if microwave dielectric heating or conventional heating is employed. Tryptic digests of three proteins of different size (bovine serum albumin, cytochrome c and β-casein) were thus performed at 37 °C and 50 °C using both microwave and conventional heating applying accurate internal fiber-optic probe reaction temperature measurements. The impact of the heating method on protein degradation and peptide fragment generation was analyzed by SDS-PAGE and MALDI-TOF-MS. Time-dependent tryptic digestion of the three proteins and subsequent analysis of the corresponding cleavage products by MALDI-TOF provided virtually identical results for both microwave and conventional heating. In addition, the impact of electromagnetic field strength on the tertiary structure of trypsin and BSA was evaluated by molecular mechanics calculations. These simulations revealed that the applied field in a typical laboratory microwave reactor is 3–4 orders of magnitude too low to induce conformational changes in proteins or enzymes.

Structural and immunologic characterization of bovine, horse, and rabbit serum albumins

Serum albumin (SA) is the most abundant plasma protein in mammals. SA is a multifunctional protein with extraordinary ligand binding capacity, making it a transporter molecule for a diverse range of metabolites, drugs, nutrients, metals and other molecules. Due to its ligand binding properties, albumins have wide clinical, pharmaceutical, and biochemical applications. Albumins are also allergenic, and exhibit a high degree of cross-reactivity due to significant sequence and structure similarity of SAs from different organisms. Here we present crystal structures of albumins from cattle (BSA), horse (ESA) and rabbit (RSA) sera. The structural data are correlated with the results of immunological studies of SAs. We also analyze the conservation or divergence of structures and sequences of SAs in the context of their potential allergenicity and cross-reactivity. In addition, we identified a previously uncharacterized ligand binding site in the structure of RSA, and calcium binding sites in the structure of BSA, which is the first serum albumin structure to contain metal ions.

Chitosan–bovine serum albumin complex formation: A model to design an enzyme isolation method by polyelectrolyte precipitation

Interactions between a model protein (bovine serum albumin—BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan–BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.

Encapsulation of BSA in polylactic acid–hyperbranched polyglycerol conjugate nanoparticles: preparation, characterization, and release kinetics

Nanoparticles based on an amphiphilic copolymer with polylactic acid (PLA) grafted onto hyperbranched polyglycerol (HPG) were prepared by the use of BSA as a model protein. The characteristics of the nanoparticles were evaluated using particle size analyzer, transmission electron microscopy, and X-ray photoelectron spectroscopy. The secondary structure of BSA released from nanoparticles were analysed by circular dichroism experiments. Cell viability of nanoparticles was also evaluated by using NIH 3T3 cells. The mechanism of BSA release was studied by fitting experimental data to three model equations. Results indicated that BSA diffusion and the polymeric relaxation jointly governed the overall release process. The detailed analysis of BSA release was performed using the first-order kinetic model equation, which gave a good fit to the experimental release data. The influence of different copolymer structures and BSA loading capacities on release profiles were also evaluated for the potential of using nanoparticles as controlled release protein delivery systems.

Radiation synthesis and characterization of protein stabilized gold nanoparticles

Gold nanoparticles were synthesized in a single step and a facile procedure by γ-irradiation using bovine serum albumin protein as a stabilizer. The results of UV–vis absorption spectroscopy, transmission electron microscopy and X-ray diffraction analyses indicated that spherical well-dispersed gold nanoparticles ranging from 2 to 7 nm were generated, depending on the irradiation doses. The conjugation of BSA to the gold nanoparticles was indicated using FTIR spectroscopy and dynamic light scattering. Moreover, the effect of irradiation on the BSA structure in the presence and absence of Au 3+ was studied by SDS-PAGE analysis. It became evident that gold nanoparticles formation partially protected against the degradation and aggregation of BSA. Methyl tetrazolium (MTT) assay demonstrated that the generated gold nanoparticles are not cytotoxic to mammalian cells in cultures.

A steady‐state and time‐resolved fluorescence, circular dichroism study on the binding of myricetin to bovine serum albumin

The binding mechanism of myricetin (Myr) to bovine serum albumin was investigated by using steady-state and time-resolved fluorescence and circular dichroism. The results of the steady-state fluorescence quenching experiment indicate that it is a static quenching process (C(Myr)/C(BSA) < or = 3) at low quencher concentration and the binding site is located near the Trp212 residue. The association constants at the different temperatures were calculated. From the thermodynamic parameters, enthalpy change (DeltaH(0)), Gibbs free energy change (DeltaG(0)) and entropy change (DeltaS(0)) obtained in the experiment, it was found that hydrophobic and electrostatic interactions play important roles in binding Myr to BSA. According to the Föster energy transfer theory, the separation distance, r, the energy transfer efficiency, E, and Föster radius, R(0), were calculated. The results obtained from the above experiments indicate that Myr can be tightly bound to BSA. Then, the effects of ionic strength, metal ion, pH and surfactants on the binding Myr and BSA were investigated, which also showed that electrostatic and hydrophobic interactions play a major role in the association process. On the other hand, the conformation and secondary structure of BSA were further studied through circular dichroism and fluorescence synchronous spectra. It was found that the conformation and secondary structure of BSA had also changed after interaction with Myr. The time-resolved fluorescence study showed that the short lifetime of BSA decreased after the addition of Myr, which implies that the buried Trp 212 is the main binding site.

Preparation of alginate coated chitosan microparticles for vaccine delivery

BackgroundAbsorption of antigens onto chitosan microparticles via electrostatic interaction is a common and relatively mild process suitable for mucosal vaccine. In order to increase the stability of antigens and prevent an immediate desorption of antigens from chitosan carriers in gastrointestinal tract, coating onto BSA loaded chitosan microparticles with sodium alginate was performed by layer-by-layer technology to meet the requirement of mucosal vaccine.ResultsThe prepared alginate coated BSA loaded chitosan microparticles had loading efficiency (LE) of 60% and loading capacity (LC) of 6% with mean diameter of about 1 μm. When the weight ratio of alginate/chitosan microparticles was greater than 2, the stable system could be obtained. The rapid charge inversion of BSA loaded chitosan microparticles (from +27 mv to -27.8 mv) was observed during the coating procedure which indicated the presence of alginate layer on the chitosan microparticles surfaces. According to the results obtained by scanning electron microscopy (SEM), the core-shell structure of BSA loaded chitosan microparticles was observed. Meanwhile, in vitro release study indicated that the initial burst release of BSA from alginate coated chitosan microparticles was lower than that observed from uncoated chitosan microparticles (40% in 8 h vs. about 84% in 0.5 h). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) assay showed that alginate coating onto chitosan microparticles could effectively protect the BSA from degradation or hydrolysis in acidic condition for at least 2 h. The structural integrity of alginate modified chitosan microparticles incubated in PBS for 24 h was investigated by FTIR.ConclusionThe prepared alginate coated chitosan microparticles, with mean diameter of about 1 μm, was suitable for oral mucosal vaccine. Moreover, alginate coating onto the surface of chitosan microparticles could modulate the release behavior of BSA from alginate coated chitosan microparticles and could effectively protect model protein (BSA) from degradation in acidic medium in vitro for at least 2 h. In all, the prepared alginate coated chitosan microparticles might be an effective vehicle for oral administration of antigens.

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.