Isoelectric Point Of Protein Research Articles

Comparative study on emulsifying and physico-chemical properties of bovine and camel acid and sweet wheys

Main goal of this research was to examine the emulsifying properties of acid and sweet wheys extracted from bovine and camel fresh milks after heating at 70 and 90 °C for 30 min at laboratory scale. Specifically, emulsifying properties (emulsification stability (ESI) and activity (EAI) indexes) and the physico-chemical characteristics (surface hydrophobicity, ζ-potential, interfacial tension and denaturation rate) of wheys were assessed. Maximum EAI and ESI were found for sweet wheys (EAI~ 2 m2/g; ESI~ 65%), with higher EAI values for the camel whey. This behavior was explained by the strongest electrostatic-repulsive forces between oil droplets under conditions away from the isoelectric-point of proteins in agreement with the ζ-potential measurements.Findings indicate that heating affected the physico-chemical properties of camel and bovine whey proteins in acidic conditions by increasing surface hydrophobicity and the ability to reduce the interfacial tension. These results confirmed the protein-protein aggregation of heated acid wheys as proved by electrophoreses.

Performance of tetraalkylammonium-based ionic liquids as constituents of aqueous biphasic systems in the extraction of ovalbumin and lysozyme

Ionic-liquid-based aqueous biphasic systems (IL-based ABS) have been described as promising platforms for the extraction and separation of proteins. However, imidazolium-based ILs have been the preferred choice, which may raise some biocompatibility and biodegradability concerns. In this work, novel ABS composed of tetraalkylammonium-based ILs and potassium phosphate solutions at different pH values (pH = 7, 8, 9 and 13, using K2HPO4/KH2PO4 or K3PO4) were investigated in terms of extraction efficiency and recovery yield for two proteins, namely ovalbumin and lysozyme. These proteins were selected due to their wide application in several sectors, being present in egg white. At pH 7, the complete extraction and recovery of lysozyme to the IL-rich phase are achieved in all systems; however, low recovery yields of ovalbumin are obtained with ABS formed by ILs with longer alkyl side chains. Furthermore, an increase in the pH above the proteins isoelectric point is deleterious for their recovery in the IL-rich phase. In order to characterize the molecular-level mechanisms that could maximize the proteins recovery, molecular docking studies were carried out, showing that ILs that preferentially establish hydrophobic interactions with these proteins are those that lead to their aggregation and lower recovery yields. Finally, it is shown the proteins recovery from the IL-rich phase by ice cold ethanol precipitation, where up to 99% of lysozyme can be recovered. These results support the viability of adequate IL-based ABS to extract ovalbumin and lysozyme and the possibility of recovering stable proteins from the IL-rich phase into an adequate buffered aqueous solution, thus contributing to the design of effective separation processes.

Localization-specific distributions of protein pI in human proteome are governed by local pH and membrane charge

BackgroundWhole-proteome distributions of protein isoelectric point (pI) values in different organisms are bi- or trimodal with some variations. It was suggested that the observed multimodality of the proteome-wide pI distributions is associated with subcellular localization-specific differences in the local pI distributions. However, the factors responsible for variation of the intracellular localization-specific pI profiles have not been investigated in detail.ResultsIn this work, we explored proteome-wide pI distributions of 32,138 human proteins predicted to reside in 10 subcellular compartments, as well as the pI distributions of experimentally observed lysosomal and Golgi proteins. The distributions were found to differ significantly, although all of them adhered to the major recurrent bimodal pattern. Grossly, acid-biased and alkaline-biased patterns with various minor statistical features were observed at different subcellular locations. Bioinformatics analysis revealed the existence of strong statistically significant correlations between protein pI and subcellular localization. Most markedly, protein pI was found to correlate positively with nuclear and mitochondrial locations and negatively with cytoskeletal, cytoplasmic, lysosomal and peroxisomal environment. Further analysis demonstrated that subcellular compartment-specific pI distributions are greatly influenced by local pH and organelle membrane charge. Multiple nonlinear regression analysis identified a polynomial function of the two variables that best fitted the mean pI values of the localization-specific pI distributions. A high coefficient of determination calculated for this regression (R2 = 0.98) suggests that local pH and organelle membrane charge are the major factors responsible for variation of the intracellular localization-specific pI profiles.ConclusionsOur study demonstrates that strong correlations exist between protein pI and subcellular localization. The specific pI distributions at different subcellular locations are defined by local environment. Predominantly, it is the local pH and membrane charge that shape the organelle-specific protein pI patterns. These findings expand our understanding of spatial organization of the human proteome.

Encapsulation of Lutein in Nanoemulsions Stabilized by Resveratrol and Maillard Conjugates.

Lutein is incorporated into foods as a natural yellow pigment and nutraceutical. The introduction of lutein into many foods and beverages, however, is problematic because of its strong hydrophobicity and poor chemical stability. In this research, lutein-loaded nanoemulsions were prepared to overcome this problem. Casein-dextran Maillard conjugates or physical complexes were utilized as emulsifiers, while either medium chain triglycerides (MCT) or grape seed oil (GSO) were used as carrier oils. The impact of resveratrol addition on nanoemulsion stability was also examined. The influence of storage temperature, pH, and CaCl2 concentration on the chemical and physical stability of the nanoemulsions was measured. The casein-dextran conjugates were highly effective at improving the physical resistance of the nanoemulsions to environmental stresses, but had a detrimental effect on their color stability. Conversely, nanoemulsions prepared from casein-dextran physical complexes were unstable around the protein's isoelectric point (pH 4.6), as well as upon addition of CaCl2 . Incorporation of resveratrol and GSO into the nanoemulsions decreased lutein degradation and color fading at all temperatures. This study shows that casein-dextran conjugates are highly effective at improving the physical stability of lutein-loaded nanoemulsions, while resveratrol and GSO are effective at improving their chemical stability. PRACTICAL APPLICATION: Lutein can be used by the food industry to create "clean label" and functional food products. The major challenges in incorporating lutein in foods are its poor chemical stability and its high hydrophobicity, which makes it difficult to incorporate. Emulsion-based delivery systems assembled from natural ingredients may address these challenges. In this study, the impact of Maillard conjugates fabricated from caseinate and dextran, as well as resveratrol addition, on the formation and stability of lutein-enriched nanoemulsions was determined. The information obtained from this study will help the formulation of more effective functional foods and beverage products.

Isoelectric Points of Proteins at the Air/Liquid Interface and in Solution.

Electrostatic interactions play essential roles in determining the function, colloidal stability, and adsorption of proteins on different surfaces and interfaces. Therefore, a molecular-level understanding of the charge state of the proteins under different conditions is required to explain their macroscopic properties. In this study, we have employed an inherently surface-sensitive spectroscopic tool, sum frequency generation spectroscopy, to determine the charge state of a wide range of proteins as a function of pH at the air/liquid interface via measurement of the degree of orientation of water molecules. We compared the isoelectric point (IEP) of the 12 investigated proteins at the air/liquid interface with that in the bulk solution obtained through zeta potential measurements. Ellipsometry is performed to determine the film thickness at the air/liquid interface at different charge states. In particular, protein aggregation at the IEP is reflected by increased film thickness. For all proteins, the interfacial point of zero charge is close (with less than 1 pH unit variation) to that in the bulk solution.

Design rules for encapsulating proteins into complex coacervates.

We investigated the encapsulation of the model proteins bovine serum albumin (BSA), human hemoglobin (Hb), and hen egg white lysozyme (HEWL) into two-polymer complex coacervates as a function of polymer and solution conditions. Electrostatic parameters such as pH, protein net charge, salt concentration, and polymer charge density can be used to modulate protein uptake. While the use of a two-polymer coacervation system enables the encapsulation of weakly charged proteins that would otherwise require chemical modification to facilitate electrostatic complexation, we observed significantly higher uptake for proteins whose structure includes a cluster of like-charged residues on the protein surface. In addition to enhancing uptake, the presence of a charge patch also increased the sensitivity of the system to modulation by other parameters, including the length of the complexing polymers. Lastly, our results suggest that the distribution of charge on a protein surface may lead to different scaling behaviour for both the encapsulation efficiency and partition coefficient as a function of the absolute difference between the protein isoelectric point and the solution pH. These results provide insight into possible biophysical mechanisms whereby cells can control the uptake of proteins into coacervate-like granules, and suggest future utility in applications ranging from medicine and sensing to remediation and biocatalysis.

СТРУКТУРНАЯ ИЗМЕНЧИВОСТЬ ГЕНА БОГАТОГО МЕТИОНИНОМ АЛЬБУМИНА SFA8 ПОДСОЛНЕЧНИКА

Background. The 2S albumins of sunflower and other oilseed plants possess a high nutritional quality, the defense activity against fungi diseases casual gents and also valuable functional properties. The major component of albumin fraction, the SFA8 protein consists of 103 amino acid residues among which methionine constitutes 15 Mole %. In the cultivated sunflower gene pool the SFA8 structural gene is represented by the two alleles the products of which have different isoelectric points and differ by the electrophoretic mobility, however molecular mechanisms of the polymorphism are still unknown. Results. The amplified sequences of the SFA8 gene from seven Helianthus annuus L. accessions and three accessions of wild Helianthus L. species from VIR collection were sequences. The intron of 258-303 bp length depending on the genotype was firstly found in the central part of the gene. The length of the first exon constitutes 99 bp, the second exon is of 210 bp length. The nucleotide and translated amino acid sequences are polymorphic among different genotypes. The line VIR 130 in which the two expressing SFA8 proteins, the normal polypeptide with isoelectric point (pI) approximately 6.0 (normal SFA8) and its allelic variant with pI 6.5 (variant SFA8) have been earlier revealed possesses two types of the SFA8 encoding sequence. In one sequence the substitution 108С—G is present that results in the substitution of the polar uncharged amino acid serine for the positively charged arginine and respectively in alteration of the protein charge and isoelectric point. The intron sequence is also polymorphic and characterized by the presence of indels of approximately 45 bp. The intron sequences of all accessions contain dinucleotides GT at the 5΄ end and AG at the 3΄ end which are characteristic for consensus sequences of splicing sites in the U2-type introns. The variants of the secondary structure of the SFA8 intron sequences of H. argophyllus Torr. & A. Gray and all the analyzed H. annuus genotypes are similar and differ from those of H. petiolaris Nutt. and H. giganteus L. Conclusions. The data on the SFA8 gene sequence polymorphism are important understanding the molecular mechanisms of genotypic differences in biochemical and functional properties of the protein, and he revealed differences in the intron secondary structure can be important for understanding expression patterns of the protein.

Native Polyacrylamide Gels.

Proteins can easily be separated by polyacrylamide gel electrophoresis (PAGE) in the presence of a detergent and under (heat-) denaturing and (non- or) reducing conditions. The most commonly used detergent is sodium dodecyl sulfate (SDS). The major function of SDS is to shield the respective charge of the proteins present in the mixture to be analyzed and to provide all proteins with a negative charge. As a consequence, the proteins will be separated according to their molecular weight. Electrophoresis of proteins can also be performed in the absence of SDS. Using such "native" conditions, the charge of each of the proteins, which will depend on the primary amino acid sequence of the protein (isoelectric point) and the pH during electrophoresis, will mainly influence the mobility of the respective protein during electrophoresis. Here we describe a starting protocol for "native" PAGE.

Computational analysis of the amino acid interactions that promote or decrease protein solubility

The solubility of globular proteins is a basic biophysical property that is usually a prerequisite for their functioning. In this study, we probed the solubility of globular proteins with the help of the statistical potential formalism, in view of objectifying the connection of solubility with structural and energetic properties and of the solubility-dependence of specific amino acid interactions. We started by setting up two independent datasets containing either soluble or aggregation-prone proteins with known structures. From these two datasets, we computed solubility-dependent distance potentials that are by construction biased towards the solubility of the proteins from which they are derived. Their analysis showed the clear preference of amino acid interactions such as Lys-containing salt bridges and aliphatic interactions to promote protein solubility, whereas others such as aromatic, His-π, cation-π, amino-π and anion-π interactions rather tend to reduce it. These results indicate that interactions involving delocalized π-electrons favor aggregation, unlike those involving no (or few) dispersion forces. Furthermore, using our potentials derived from either highly or weakly soluble proteins to compute protein folding free energies, we found that the difference between these two energies correlates better with solubility than other properties analyzed before such as protein length, isoelectric point and aliphatic index. This is, to the best of our knowledge, the first comprehensive in silico study of the impact of residue-residue interactions on protein solubility properties.The results of this analysis provide new insights that will facilitate future rational protein design applications aimed at modulating the solubility of targeted proteins.

Surface modification of protein enhances encapsulation in chitosan nanoparticles

Chitosan nanoparticles have a huge potential as nanocarriers for environmental and biomedical purposes. Protein encapsulation in nano-sized chitosan provides protection against inactivation, proteolysis, and other alterations due to environmental conditions, as well as the possibility to be targeted to specific tissues by ligand functionalization. In this work, we demonstrate that the chemical modification of the protein surface enhances the protein loading in chitosan nanocarriers. Encapsulation of green fluorescent protein and the cytochrome P450 was studied. The increase of electrostatic interactions between the free amino groups of chitosan and the increased number of free carboxylic groups in the protein surface enhance the protein loading, protein retention, and, thus, the enzymatic activity of chitosan nanoparticles. The chemical modification of protein surface with malonic acid moieties reduced drastically the protein isoelectric point increasing the protein interaction with the polycationic biomaterial and chitosan. The chemical modification of protein does not alter the morphology of chitosan nanoparticles that showed an average diameter of 18 nm, spheroidal in shape, and smooth surfaced. The strategy of chemical modification of protein surface, shown here, is a simple and efficient technique to enhance the protein loading in chitosan nanoparticles. This technique could be used for other nanoparticles based on polycationic or polyanionic materials. The increase of protein loading improves, doubtless, the performance of protein-loaded chitosan nanoparticles for biotechnological and biomedical applications.

Association Constants in the Bovine Serum Albumin/Human Serum Albumin–Tween 20 System in Aqueous Solutions

The association constants in the bovine serum albumin/human serum albumin–Tween 20 system are determined in aqueous solutions at pH 3.5–8.0 by means of nonpolarized fluorescence and protein fluorescence quenching. It is found that the most efficient association of serum albumin molecules with Tween 20 micelles occurs at pH 5.0 near the isoelectric points of proteins.

Activity of non-canonical pluripotency-associated transcription factors in goat cumulus-oocyte complexes

Pluripotency-associated transcription factors (PATFs) are involved in several cellular processes, such as transcription initiation, cellular metabolism, and cell fate determination in preimplantation embryos. Despite growing understanding of PATFs in pluripotent human and mouse cells, their roles in other species or cell types remain limited. The work aimed to determine the transcript availability of OCT4 and four non-canonical PATFs (NAC1, NR0B1, RONIN, and ZFP281) in goat ovaries, eggs, and cumulus cells. Goat ovaries were collected at slaughterhouses and cumulus-oocyte complexes (COC) were retrieved from 3 to 6 mm follicles. The COCs were subject to in vitro maturation in TCM-199 medium containing 2 mM l-glutamine, 5.0 μg mL−1 FSH, 10% FBS, and antibiotics at 38.5 °C with saturated humidity and 5% CO2 during 24 h. Gene activity was detected by both conventional reverse transcription quantitative polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR). In silico analysis were carried out for protein physical properties, phenetic analysis, and protein domains within goat, mouse, and human PATF orthologs. Products of all five PATFs analyzed (NAC1, NR0B1, OCT4, RONIN, and ZFP281) were detected in goat eggs, but NR0B1 was not detected in the ovary. Surprisingly, NR0B1, RONIN, and ZFP281 were also expressed in cumulus cells. All PATFs and their binding partners (i.e., OCT4-SOX2, RONINHCF1, and NANOG-NR0B1-ZFP281-NAC1) showed similar protein size, molecular weight, and isoelectric point among species. The phenetic analysis clustered more closely goat and human proteins for OCT4, SOX2, NANOG, and NR0B1. Finally, both DNA-binding and transactivation domains were also highly conserved among species, particularly between goats and humans, suggesting that these PATFs may have conserved functions and similar target genes. In conclusion, non-canonical PATFs have distinctive transcript abundance in goat eggs and cumulus cells, while in silico approaches suggest that these genes may play similar roles to their mouse and human orthologs.

Application of Response Surface Methodology in the Preparation of Pectin-Caseinate Nanocomplexes for Potential Use as Nutraceutical Formulation: A Statistical Experimental Design Analysis

Background: The formation of electrostatic complexes between two types of biopolymers, sodium Caseinate (a derivative from most abundant milk protein) and Pectin (a natural hetro polysaccharide), was studied as a function of biopolymers concentrations and pH of solutions (3.9- 4.3). Method: The size and morphology of the resulted complexes were investigated by using of laser light scattering and transmission electron microscopy, respectively. Response surface methodology (A three-factor, three levels Box-Behnken design) was used for the optimization procedure with pH, pectin and sodium Caseinate concentrations as independent variables. Particle size and polydispersity index of nanocomplexes were considered as dependent variables. Results: Negatively charged nanocomplexes were produced below the isoelectric point of protein (5.4), at pH 4.1 with a suitable colloidal stability and average particle size of about 100 nm. It was found that the particle size of nanocomplexes could be controlled by changing in variables. Conclusion: In conclusion response surface methodology are simple, rapid and beneficial approach for preparation, optimization and investigation of the effect of independent variables on the properties of products.

High protein ingredients of microalgal origin: Obtainment and functional properties

Abstract Protein concentrates and isolates were developed from the microalga Spirulina sp. LEB 18 biomass and characterized by functional aspects for food application. Proteins were solubilized at pH 11 and precipitated at pH 4.2 (protein isoelectric point determined by potentiometric titration), with the aid of a high-speed homogenizer. With this procedure, it was possible to obtain a protein concentrate with 83.9 ± 1.7 wt% of protein and a protein isolate with 91.3 ± 1.2 wt%. Protein extraction from this microalga allowed a significant increase in protein solubility and foam stability. Furthermore, proteins from both concentrate and isolate presented higher resistance to thermal denaturation than the original proteins of Spirulina sp. LEB 18 biomass. These results show the application potential of the concentrate and the protein isolate from Spirulina sp. LEB 18 in foods for protein supplementation, since they have >80 and 90% protein, respectively. Industrial relevance text Using proteins from microalgal sources can serve as a sustainable alternative to meet world food demand. The isoelectric precipitation extraction method can be applied on a large scale, achieving high yields. Protein extracts present potential application in specific foods for protein supplementation. Protein extraction can be allied to other biocomposites extraction, such as carbohydrates and lipids for biofuels production.

Enhancement of physicochemical properties of whey protein-stabilized nanoemulsions by interfacial cross-linking using cinnamaldehyde

The impact of incorporating different levels of cinnamaldehyde (CA) into the oil phase used to prepare whey protein isolate (WPI)-stabilized nanoemulsions was investigated. Stable nanoemulsions could not be formed by pure CA, which was attributed to gravitational separation, but they could be formed from mixtures of CA and triglycerides. The mean particle diameter produced by homogenization decreased with increasing CA, which was attributed to the reduction of oil phase interfacial tension and viscosity. CA enhanced the physical stability of the nanoemulsions to salt addition (0–500 mM) and thermal processing (30–90 °C), but could not prevent droplet flocculation near the protein's isoelectric point (around pH 5). SDS-PAGE and surface load measurements indicated that more protein accumulated at the droplet surfaces in the presence of CA, which was attributed to interfacial crosslinking. CA may therefore be a useful means of modulating the physicochemical properties of protein-stabilized nanoemulsions.

Self-assembled curcumin-soluble soybean polysaccharide nanoparticles: Physicochemical properties and in vitro anti-proliferation activity against cancer cells

Nanoencapsulation of lipophilic bioactive compounds in food biopolymers is important to functional beverages, but protein-based nanocapsules are unstable around the isoelectric point of protein. The objectives of this work were to study physicochemical properties of self-assembled curcumin-soluble soybean polysaccharide (SSPS) nanoparticles and evaluate the activities against proliferation of human colon HCT116 and mammary adenocarcinoma MCF-7 cancer cells before and after simulated digestions. Capsules with a hydrodynamic diameter of 200–300 nm and an encapsulation efficiency of ∼90% were self-assembled after increasing curcumin-SSPS mixture to pH 12.0 and lowering pH to 7.0. The capsule dispersions were stable at pH 2.0–7.0 and after heating at 95 °C for 1 min. No significant difference was observed for the viability of HCT 116 and MCF-7 cells challenged with 0.4, 4.0, and 40 μg/ml nanoencapsulated curcumin before and after simulated gastric and intestinal digestions. These findings may be significant to help develop functional beverages for disease prevention.

Self-Organization of Recombinant Membrane Porin OmpF from Yersinia pseudotuberculosis in Aqueous Environments.

Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8M urea (pH8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.

Cloning and characterization of F3PYC gene encoding pyruvate carboxylase in Aspergillus flavus strain (F3)

Pyruvate carboxylase is a major enzyme for biosynthesis of organic acids like; citric acid, fumeric acid, and L-malic acid. These organic acids play very important role for biological remediation of heavy metals. In this study, gene walking method was used to clone and characterize pyruvate carboxylase gene (F3PYC) from heavy metal resistant indigenous fungal isolate Aspergillus flavus (F3). 3579bp of an open reading frame which encodes 1193 amino acid protein (isoelectric point: 6.10) with a calculated molecular weight of 131.2008kDa was characterized. Deduced protein showed 90-95% similarity to those deduced from PYC gene from different fungal strains including; Aspergillus parasiticus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus clavatus, and Aspergillus niger. Protein generated from the PYC gene was a homotetramer (α4) and having four potential N-linked glycosylation sites and had no signal peptide. Amongst most possible N-glycosylation sites were -N-S-S-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, N-G-S-S- at 517 amino acid, and N-T-S-R- at 1111 amino acid, with several functions have been proposed for the carbohydrate moiety such as thermal stability, pH, and temperature optima for activity and stabilization of the three-dimensional structure. Hence, cloning of F3PYC gene from A. flavus has important biotechnological applications.

Hemoglobin bioconjugates with surface-protected gold nanoparticles in aqueous media: The stability depends on solution pH and protein properties

The identification of the factors that dictate the formation and physicochemical properties of protein-nanomaterial bioconjugates are important to understand their behavior in biological systems. The present work deals with the formation and characterization of bioconjugates made of the protein hemoglobin (Hb) and gold nanoparticles (AuNP) capped with three different molecular layers (citrate anions (c), 6-mercaptopurine (MP) and ω-mercaptoundecanoic acid (MUA)). The main focus is on the behavior of the bioconjugates in aqueous buffered solutions in a wide pH range. The stability of the bioconjugates have been studied by UV–visible spectroscopy by following the changes in the localized surface resonance plasmon band (LSRP), Dynamic light scattering (DLS) and zeta-potential pH titrations. It has been found that they are stable in neutral and alkaline solutions and, at pH lower than the protein isoelectric point, aggregation takes place. Although the surface chemical properties of the AuNPs confer different properties in respect to colloidal stability, once the bioconjugates are formed their properties are dictated by the Hb protein corona. The protein secondary structure, as analyzed by Attenuated total reflectance infrared (ATR-IR) spectroscopy, seems to be maintained under the conditions of colloidal stability but some small changes in protein conformation take place when the bioconjugates aggregate. These findings highlight the importance to keep the protein structure upon interaction with nanomaterials to drive the stability of the bioconjugates.

Bovine Serum Albumin Adsorption at a Silica Surface Explored by Simulation and Experiment.

Molecular details of BSA adsorption on a silica surface are revealed by fully atomistic molecular dynamics (MD) simulations (with a 0.5 μs trajectory), supported by dynamic light scattering (DLS), zeta potential, multiparametric surface plasmon resonance (MP-SPR), and contact angle experiments. The experimental and theoretical methods complement one another and lead to a wider understanding of the mechanism of BSA adsorption across a range of pH 3-9. The MD results show how the negatively charged BSA at pH7 adsorbs to the negatively charged silica surface, and reveal a unique orientation with preserved secondary and tertiary structure. The experiments then show that the protein forms complete monolayers at ∼ pH6, just above the protein's isoelectric point (pH5.1). The surface contact angle is maximum when it is completely coated with protein, and the hydrophobicity of the surface is understood in terms of the simulated protein conformation. The adsorption behavior at higher pH > 6 is also consistently interpreted using the MD picture; both the contact angle and the adsorbed protein mass density decrease with increasing pH, in line with the increasing magnitude of negative charge on both the protein and the surface. At lower pH < 5 the protein starts to unfold, and the adsorbed mass dramatically decreases. The comprehensive picture that emerges for the formation of oriented protein films with preserved native conformation will help guide efforts to create functional films for new technologies.