977 resultados para PROTEIN ADSORPTION


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R-phycoerythrin, a light-harvesting protein in some marine algae, and can be widely used in medicine, was isolated and purified from a red alga, Palmaria palmata (Lannaeus) Kuntze, using the streamline column (expanded bed adsorption) combined with ion-exchange chromatography. Because the crude extract was applied to the column upwardly, the column would not be blocked by polysaccharides usually very abundant in the extract of marine alga, this kind of blockage could hardly lie overcome in ordinary chromatographic column. After applying the crude extract containing 0.5 mol/L (NH4)(2)SO4, (NH4)(2)SO4 solution of different concentrations (0.2 mol/L, 0.1 mol/L and 0.05 mol/L) was used to elute the column downwardly and the eluates were collected and desalted. The desalted eluates were then applied onto all ion-exchange chromatographic column loaded with Q-sepharose for further purification of the R-phycoerythrin. Through these two steps, the purity (OD565/OD280) of the R-phycoerythrin from P. palmata was up to 3.5, more than 3.2, the commonly accepted criterion for purity, and the yield of the purified R-phycoerythrin could reach 0.122 mg/g of frozen P. palmata, much higher than that of phycobiliproteins purified with the previous methods. The result indicated that the cost of R-phycoerythrin will drop down with the method reported in this article.

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C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE (TM) Column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin. (c) 2006 Elsevier B.V. All rights reserved.

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Tangential flow affinity membrane cartridge (TFAMC) fs a new model of immunoadsorption therapy for hemoperfusion. Recombinant Protein A was immobilized on the membrane cartridge through Schiff base formation for extracorporeal IgG and immune complex removal from blood. Flow characteristics, immunoadsorption capacity and biocompatibility of protein A TFAMC were studied. The results showed that the pressure drop increased with the increasing flow rate of water, plasma and blood, demonstrating reliable strength of membrane at high now rare. The adsorption capacities of protein A TFAMC for IgG from human plasma and blood were measured. The cartridge with 139 mg protein A immobilized on the matrix (6 mg protein A/g dry matrix) adsorbed 553 mg IgG (23.8 mg IgG/g dry matrix) from human plasma and 499.4 mg IgG (21.5 mg IgG/g dry matrix) from human blood, respectively. The circulation time had a major influence on IgG adsorption capacity, but the flow rate had little influence. Experiments in vitro and in vivo confirmed that protein A TFAMC mainly adsorbed Ige and Little of other plasma proteins, and that blood cell damage was negligible. The extracorporeal circulation system is safe and reliable. Copyright (C) 1999 John Wiley & Sons, Ltd.

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Particulate colloids often occur together with proteins in sewage-impacted water. Using Bovine Serum Albumin (BSA) as a surrogate for protein in sewage, column experiments investigating the capacity of iron-oxide coated sands to remove latex microspheres from water revealed that microsphere attenuation mechanisms depended on antecedent BSA coverage. Dual pulse experiment (DPE) results suggested that where all BSA was adsorbed, subsequent multiple pore volume microsphere breakthrough curves reflected progressively reduced colloid deposition rates with increasing adsorbed BSA content. Modelling colloid responses suggested adsorption of 1 µg BSA generated the same response as blockage by between 7.1x108 and 2.3x109 deposited microspheres. By contrast, microsphere responses in DPEs where BSA coverage of the deposition sites approached/ reached saturation revealed the coated sand maintained a finite capacity to attenuate microspheres, even when incapable of further BSA adsorption. Subsequent microsphere breakthrough curves demonstrated the matrix’s colloid attenuation capacity progressively increased with continued microsphere deposition. Experimental findings suggested BSA adsorption on the sand surface approaching/ reaching saturation generated attractive deposition sites for colloids, which became progressively more attractive with further colloid deposition (filter ripening). Results demonstrate that adsorption of a single type of protein may either enhance or inhibit colloid mobility in saturated porous media.

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A methodology for discovering the mechanisms and dynamics of protein clustering on solid surfaces is presented. In situ atomic force microscopy images are quantitatively compared to Monte Carlo simulations using cluster statistics to differentiate various models. We study lysozyme adsorption on mica as a model system and find that all surface-supported clusters are mobile, not just the monomers, with diffusion constant inversely related to cluster size. The surface monomer diffusion constant is measured to be D1∼9×10-16  cm2 s-1, such a low value being difficult to measure using other techniques.

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Despite its relevance to a wide range of technological and fundamental areas, a quantitative understanding of protein surface clustering dynamics is often lacking. In inorganic crystal growth, surface clustering of adatoms is well described by diffusion-aggregation models. In such models, the statistical properties of the aggregate arrays often reveal the molecular scale aggregation processes. We investigate the potential of these theories to reveal hitherto hidden facets of protein clustering by carrying out concomitant observations of lysozyme adsorption onto mica surfaces, using atomic force microscopy. and Monte Carlo simulations of cluster nucleation and growth. We find that lysozyme clusters diffuse across the substrate at a rate that varies inversely with size. This result suggests which molecular scale mechanisms are responsible for the mobility of the proteins on the substrate. In addition the surface diffusion coefficient of the monomer can also be extracted from the comparison between experiments and simulations. While concentrating on a model system of lysozyme-on-mica, this 'proof of concept' study successfully demonstrates the potential of our approach to understand and influence more biomedically applicable protein-substrate couples.

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Colloidal gas aphrons (CGA), which are surfactant stabilised microbubbles, have been previously applied for the recovery of proteins from model mixtures and a few studies have demonstrated the potential of these dispersions for the selective recovery of proteins from complex mixtures. However there is a lack of understanding of the mechanism of separation and forces governing the selectivity of the separation. In this paper a mechanistic study is carried out to determine the main factors and forces influencing the selectivity of separation of whey proteins with CGA generated from ionic surfactants. Two different separation strategies were followed: (i) separation of lactoferrin and lactoperoxidase by anionic CGA generated from a solution of sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT); (ii) separation of beta-lactoglobulin by cationic CGA generated from a solution of cetyltrimethylammonium bromide (CTAB). Separation results indicate that electrostatic interactions are the main forces determining the selectivity however these could not completely explain the selectivities obtained following both strategies. Protein-surfactant interactions were studied by measuring the zeta potential changes on individual proteins upon addition of surfactant and at varying pH. Interestingly strongest electrostatic interactions were measured at those pH and surfactant to protein mass ratios which were optimum for protein separation. Effect of surfactant on protein conformation was determined by measuring the change in fluorescence intensity upon addition of surfactant at varying pH. Differences in the fluorescence patterns were detected among proteins which were correlated to differences in their conformational features which could in turn explain their different separation behaviour. The effect of conformation on selectivity was further proven by experiments in which conformational changes were induced by pre-treatment of whey (heating) and by storage at 4 degrees C. Overall it can be concluded that separation of proteins by ionic CGA is driven mainly by electrostatic interactions however conformational features will finally determine the selectivity of the separation with competitive adsorption having also an effect. (c) 2006 Elsevier B.V. All rights reserved.

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The surfactant properties of aqueous protein mixtures ( ranaspumins) from the foam nests of the tropical frog Physalaemus pustulosus have been investigated by surface tension, two-photon excitation. uorescence microscopy, specular neutron reflection, and related biophysical techniques. Ranaspumins lower the surface tension of water more rapidly and more effectively than standard globular proteins under similar conditions. Two- photon excitation. uorescence microscopy of nest foams treated with fluorescent marker ( anilinonaphthalene sulfonic acid) shows partitioning of hydrophobic proteins into the air-water interface and allows imaging of the foam structure. The surface excess of the adsorbed protein layers, determined from measurements of neutron reflection from the surface of water utilizing H2O/D2O mixtures, shows a persistent increase of surface excess and layer thickness with bulk concentration. At the highest concentration studied ( 0.5 mg ml(-1)), the adsorbed layer is characterized by three distinct regions: a protruding top layer of similar to20 Angstrom, a middle layer of similar to30 Angstrom, and a more diffuse submerged layer projecting some 25 Angstrom into bulk solution. This suggests a model involving self-assembly of protein aggregates at the air-water interface in which initial foam formation is facilitated by specific surfactant proteins in the mixture, further stabilized by subsequent aggregation and cross-linking into a multilayer surface complex.

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Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning.

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The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

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The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

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We describe the development of a label free method to analyze the interactions between Ca(2+) and the porcine S100A12 protein immobilized on polyvinyl butyral (PVB). The modified gold electrodes were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and surface plasmon resonance (SPR) techniques. SEM analyses of PVB and PVB-S100A12 showed a heterogeneous distribution of PVB spherules on gold surface. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due the adsorption of PVB-S100A12, and confirm the existence of a positive response of the immobilized S100Al2 to the presence of calcium ions. The biosensor exhibited a wide linear response to Ca(2+) concentrations ranging from 12.5 to 200 mM. The PVB-S100A12 seems to be bound to the gold electrode surface by physical adsorption: we observed an increase of 1184.32 m degrees in the SPR angle after the adsorption of the protein on the PVB surface (in an indication that 9.84 ng of S100A12 are adsorbed per mm(2) of the Au-PVB electrode), followed by a further increase of 581.66 m degrees after attachment of the Ca(2+) ions. In addition, no SPR response is obtained for non-specific ions. These studies might be useful as a platform for the design of new reusable and sensitive biosensing devices that could find use in the clinical applications. (C) 2010 Elsevier B.V. All rights reserved.

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Cationic supported bilayers on latex are useful to isolate and immobilize oppositely charged proteins as a monomolecular layer over a range of low protein concentrations and particle number densities. Cholera toxin (CT) from Vibrio cholerae, an 87 kDa AB(5) hexameric protein and bovine serum albumin (BSA) self-assembled on dioctadecyldimethylammonium bromide (DODAB) supported bilayers with high affinity yielding highly organized and monodisperse particulates at 5 x 10(9) particles/mL, over a range of low protein concentrations (0-0.025 mg/mL BSA or CT). Protein association onto the bilayer-covered polystyrene sulfate (PSS) was determined from adsorption isotherms, dynamic light scattering for size distributions and zeta-potential analysis revealing a monomolecular, thin and highly organized protein layer surrounding each particle with potential for biospecific recognition such as antigen-antibody, receptor-ligand, hybridization of oligonucleotide sequences, all of them important in immunodiagnosis, selective biomolecular chromatographic separations, microarrays design and others.

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Electrochemical impedance spectroscopy (EIS) in pH 6.9 phosphate buffer solution was used to investigate each step of the procedure employed to modify a screen-printed electrode (SPE). The SPE was modified with self-assembled monolayers (SAMs) of cystamine (CYS, deposited from 20 mM solution), followed by glutaraldehyde (GA, 0.3 M solution). The Trypanosoma cruzi antigen was immobilized using different deposition times. The influence of incubation time (2-18 h) of protein was also investigated. The topography of modified electrode with this protein was investigated by atomic force microscopy (AFM). Interpretation of impedance data was based on physical and chemical adsorption, and degradation of the layer at high and meddle frequencies, and charge transfer reaction involving mainly the reduction of oxygen at low frequencies. EIS studies on modified electrodes with Tc85 protein immobilized for different incubation times indicated that the optimum incubation time was 6-8 h. It was demonstrated that EIS is a good technique to evaluate the different steps and the integrity of the surface modifications, and to optimize the incubation time of protein in the development of biosensors. (C) 2010 Elsevier B.V. All rights reserved.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)