In vitro hydroxyapatite adsorbed salivary proteins

In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified.     

Monte Carlo simulation of diffusion of adsorbed proteins

We present the results of three-dimensional lattice Monte Carlo simulations of protein diffusion on the liquid-solid interface in a wide temperature range including the most interesting temperatures (from slightly below T(f) and up to T(c), where T(f) and T(c) are the folding and collapse temperatures). For the model under consideration (27 monomers of two types), the temperature dependence of the diffusion coefficient is found to obey the Arrhenius law with the normal value (approximately 10(-2)-10(-3) cm(2)/s) of the preexponential factor. Proteins 2000;39:76-81.     

Monte Carlo simulation of denaturation of adsorbed proteins

Denaturation of model proteinlike molecules at the liquid–solid interface is simulated over a wide temperature range by employing the lattice Monte Carlo technique. Initially, the molecule containing 27 monomers of two types (A and B) is assumed to be adsorbed in the native folded state (a 3 × 3 × 3 cube) so that one of its sides is in contact with the surface. The details of the denaturation kinetics are found to be slightly dependent on the choice of the side, but the main qualitative conclusions hold for all the sides. In particular, the kinetics obey approximately the conventional first-order law at T > T_c (T_c is the collapse temperature for solution). With decreasing temperature, below T_c but above T_f (T_f is the folding temperature for solution), deviations appear from the first-order kinetics. For the most interesting temperatures, that is, below T_f, the denaturation kinetics are shown to be qualitatively different from the conventional ones. In particular, the denaturation process occurs via several intermediate steps due to trapping in metastable states. Mathematically, this means that (i) the transition to the denatured state of a given molecule is nonexponential, and (ii) the denaturation process cannot be described by a single rate constant k_r. One should rather introduce a distribution of values of this rate constant (different values of k_r correspond to the transitions to the altered state via different metastable states). Proteins 30:168–176, 1998. © 1998 Wiley-Liss, Inc.     

Mobility of adsorbed proteins: a Brownian dynamics study

We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies.     

Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports

The stabilization achieved by different immobilization protocols have been compared using three different enzymes (glutaryl acylase (GAC), D-aminoacid oxidase (DAAO), and glucose oxidase (GOX)): adsorption on aminated supports, treatment of this adsorbed enzymes with glutaraldehyde, and immobilization on glutaraldehyde pre-activated supports. In all cases, the treatment of adsorbed enzymes on amino-supports with glutaraldehyde yielded the higher stabilizations: in the case of GOX, a stabilization over 400-fold was achieved. After this treatment, the enzymes could no longer be desorbed from the supports using high ionic strength (suggesting the support-protein reaction). Modification of the enzymes immobilized on supports that did not offer the possibility of react with glutaraldehyde showed the same stability that the non modified preparations demonstrating that the mere chemical modification did not have effect on the enzyme stability. This simple strategy seems to permit very good results in terms of immobilization rate and stability, offering some advantages when compared to the immobilization on glutaraldehyde pre-activated supports.     

Secondary structure of proteins adsorbed onto or embedded in polyelectrolyte multilayers

The structural changes of bovine serum albumin (BSA) and hen egg white lysozyme (HEL) upon their adsorption onto the surface or their embedding into the interior of poly(allylamine hydrochloride)-(poly(styrenesulfonate) (PAH-PSS) multilayer architectures were investigated by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The presence of the polyelectrolytes seems, as previously observed for fibrinogen (J. Phys. Chem. B 2001, 105, 11906-11916), to prevent intermolecular interactions and, thus, protein aggregation at ambient temperature. The secondary structure of the proteins was somewhat altered upon adsorption onto the polyelectrolyte multilayers. The structural changes were larger when the charges of the multilayer outer layer and the protein were opposing. The adsorption of further polyelectrolyte layers onto protein-terminated architectures (i.e., embedding the proteins into a polyelectrolyte multilayer) did not cause considerable further changes in their secondary structures. The capacity of the polyelectrolyte architectures to delay the formation of intermolecular beta-sheets upon increasing temperatures was not uniform for the studied proteins. PSS in contact with HEL could largely prevent the heat-induced aggregation of HEL. In contrast, PAH had hardly any effect on the aggregation of BSA. The differences are explained on the basis of protein-polyelectrolyte interactions, affected mostly by the nature and the strength of the ionic interactions between the polyelectrolyte-protein contact surfaces.     

Evaluation of desorption of proteins adsorbed to hydrophilic surfaces by two-dimensional electrophoresis

The evaluation of the plasma protein adsorption patterns of superparamagnetic iron oxide (SPIO) particles is of high interest concerning their in vivo fate and is carried out by two-dimensional electrophoresis (2-DE). The sample preparation is of great importance, especially the removal of the adsorbed proteins (desorption) from the particle surface for subsequent analysis by 2-DE. The removal is carried out by a desorption solution. In this study, negatively and positively charged SPIO model particles were under investigation concerning the desorption of proteins adsorbed on their surfaces. Firstly, the desorption process was determined quantitatively using the Bradford protein assay. Secondly, the removable or nonremovable protein species, from particles surface were under investigation by 2-DE. Looking at the desorption in a quantitative manner with the Bradford assay, the desorption efficacy from negatively charged particles was about 90%. In the case of the positively charged particles, the desorption efficacy seemed to be reduced, approximately 34% of the proteins remained on the surface. Comparing the protein patterns of the particles evaluated by 2-DE in the desorption solution and the proteins remaining on the particles, they confirmed the results from the protein quantification. After desorption, the IgG gamma-chains were found to be the dominant protein fraction remaining on the negatively charged particles. On the positively charged particles, many more protein species were found after desorption. The more basic the protein fragments, the more ineffective was the desorption from the positively charged model particle, and vice versa. Nevertheless, all protein spots were found qualitatively in the desorption solution, especially when the desorption solutions still containing the particles were used for the 2-DE analysis. In conclusion, 2-DE could be confirmed as the "gold standard" for determining the plasma protein adsorption patterns of nanoparticulate systems.     

Proteomic analysis of bone proteins adsorbed onto the surface of titanium dioxide

Osseointegration is the structural and functional connection between bone tissues and implants such as titanium dioxide (TiO₂). The bone-TiO₂ interface is thought to contain proteoglycans. However, exhaustive analysis of the proteins in this layer has not been performed. In this study, we evaluated the bone protein adhered on the surface of TiO₂ comprehensively. Pig bone protein was extracted by sequential elutions with guanidine, 0.1 M EDTA, and again with guanidine. The proteins obtained from these extractions were allowed to adhere to an HPLC column packed with TiO₂ and were eluted with 0.2 M NaOH. The eluted proteins were identified by LC/MS/MS and included not only proteoglycans but also other proteins such as extracellular matrix proteins, enzymes, and growth factors. Calcium depositions were observed on TiO₂ particles incubated with bone proteins, guanidine-extracted proteins adhered to TiO₂ displayed significantly high amounts of calcium depositions.     

Proteome analysis of rat serum proteins adsorbed onto synthetic octacalcium phosphate crystals

The present study was designed to determine which proteins are selectively adsorbed onto two bone substitute materials, octacalcium phosphate (OCP) and hydroxyapatite (HA) crystals, from rat serum by proteome analysis. Ground crystals of synthetic OCP and commercially available sintered HA, with the same surface area, were incubated in rat serum proteins at 37 °C for 24 h. The proteins from the crystals extracted with guanidine–HCl–EDTA were listed on the basis of the results of liquid chromatography tandem mass spectrometry (LC/MS/MS). A total of 138 proteins were detected from OCP; 103 proteins were detected from HA. Forty-eight proteins were from both crystals. A quantitative analysis of the proteins detected was performed for the extracted two bone formation-related proteins apolipoprotein E (Apo E), a protein known to promote osteoblast differentiation, and complement 3 (C3). HA adsorbed C3 (3.98 ± 0.03 fmol/μg protein) more than OCP (1.81 ± 0.07 fmol/μg protein) did, while OCP adsorbed Apo E (2.42 ± 0.03 fmol/μg protein) more than HA (1.21 ± 0.01 fmol/μg protein) did even after deleting the high-abundance proteins, such as albumin. The results demonstrated that OCP exhibits a similar property but distinct capacity with HA in adsorbing bone formation-related proteins from the serum constituents.     

Secondary structures of proteins adsorbed onto aluminum hydroxide: infrared spectroscopic analysis of proteins from low solution concentrations

Comparative studies of the secondary structures of six model proteins, adsorbed onto aluminum hydroxide gel (Alhydrogel) or in aqueous solution, were carried out by Fourier transform infrared (FTIR) spectroscopy. The analysis of high-quality spectra of all six model proteins, with a broad range of secondary structure compositions, obtained at 15 mg/ml by the conventional method and at 0.5 and 1.0 mg/ml adsorbed to Alhydrogel revealed that adsorption onto hydrophilic surfaces of aluminum hydroxide particles did not alter the secondary structures of the proteins. The results of this study suggest that adsorbing proteins to Alhydrogel provides a means of obtaining FTIR spectra to study secondary structure and conformational changes of proteins in aqueous solution at very low concentrations. The new procedure effectively lowers the concentration requirement for FTIR studies of proteins in aqueous solutions by at least 40-fold, as compared with the conventional FTIR method. It permits FTIR study of proteins to be carried out in the same concentration range as is used for circular dichroism and fluorescence, thereby making it possible to compare structural information obtained by three commonly used techniques in protein biophysical characterization.     

Modification of liposomes with N-substituted polyacrylamides: identification of proteins adsorbed from plasma

Liposomes prepared from DMPC (80%) and cholesterol (20%) were modified with a series of hydrophobically modified N-substituted polyacrylamides, namely, poly[N-isopropylacrylamide] (PNIPAM), poly[N,N-bis(2-methoxyethyl) acrylamide] (PMEAM), and poly[(3-methoxypropyl)acrylamide] (PMPAM). The hydrophobic group, N-[4-(1-pyrenylbutyl)-N-n-octadecylamine was attached to one end of the polymer chains to serve as an anchor for incorporation into the liposome bilayer. Liposome-polymer interactions were confirmed using fluorescence spectroscopy and chemical analysis. Microscopy revealed differences in aggregation tendency between unmodified and polymer-modified liposomes. Proteins adsorbed to liposome surfaces during exposure to human plasma were identified by immunoblot analysis. It was found that both unmodified and polymer-modified liposomes adsorb a wide variety of plasma proteins. Contact phase coagulation proteins, complement proteins, cell-adhesive proteins, serine protease inhibitors, plasminogen, antithrombin III, prothrombin, transferrin, alpha(2)-microglobulin, hemoglobin, haptoglobin and beta-lipoprotein as well as the major plasma proteins were all detected. Some differences were found between the unmodified and polymer-modified liposomes. The unmodified liposomes adsorbed plasminogen mainly as the intact protein, whereas on the modified liposomes plasminogen was present in degraded form. Also, the liposomes modified with PNIPAM in its extended conformation (below the lower critical solution temperature) appeared to adsorb less protein than those containing the 'collapsed' form of PNIPAM (above the LCST).     

Chromatography of proteins on columns of polyvinylpolypyrrolidone using adsorbed textile dyes as affinity ligands

A simple and inexpensive chromatography system for proteins is introduced. When the amino derivatives of chlorotriazine dyes or other azo dyes were added to an aqueous slurry of the crosslinked polymer polyvinylpolypyrrolidone they were adsorbed, thus forming an immobilized dye chromatographic matrix. The association of the textile dyes with polyvinylpolypyrrolidone did not prevent them from acting as affinity ligands for proteins. Parameters such as ionic strength, dye concentration, and column size modulated the affinity effect exerted by the immobilized dyes. Lysozyme present in an egg white protein mixture bound to a column onto which the amino derivative of Procion Brown H-A was adsorbed and was eluted with a linear gradient of KCl. The resulting purification of the enzyme was 37-fold with 80% of the original activity being recovered. Free dye eluting with the lysozyme was removed on a column of polyvinylpolypyrrolidone equilibrated with 0.5 M KCl. After chromatography, the dye column was regenerated with 0.5 M NaOH and recharged with dye. The system presented here allows one to initially screen large numbers of potentially useful protein ligands to optimize a protein separation, followed by scaleup to a system size determined by the user.     

Ordering microtubules

How do cells order their cytoplasm? While microtubule organizing centers have long been considered essential to conferring order by virtue of their microtubule nucleating activity, attention has currently refocused on the role that microtubule motors play in organizing microtubules. An intriguing set of recent findings⁽¹⁾ reveals that cell fragments, lacking microtubule organizing centers, rapidly organize microtubules into a radial array during organelle transport driven by the microtubule motor, cytoplasmic dynein. Further, interaction of radial microtubules with the cell surface centers the array, revealing that centering information resides not with centrosomes but with organized microtubules.     

Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size

Nanoparticle biological activity, biocompatibility and fate can be directly affected by layers of readily adsorbed host proteins in biofluids. Here, we report a study on the interactions between human blood plasma proteins and nanoparticles with a controlled systematic variation of properties using (18)O-labeling and LC-MS-based quantitative proteomics. We developed a novel protocol to both simplify isolation of nanoparticle bound proteins and improve reproducibility. LC-MS analysis identified and quantified 88 human plasma proteins associated with polystyrene nanoparticles consisting of three different surface chemistries and two sizes, as well as, for four different exposure times (for a total of 24 different samples). Quantitative comparison of relative protein abundances was achieved by spiking an (18)O-labeled "universal" reference into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantification across the entire sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive patterns that classified the nanoparticles based on their surface properties and size. In addition, temporal data indicated that the formation of the stable protein corona was at equilibrium within 5 min. The comprehensive quantitative proteomics results obtained in this study provide rich data for computational modeling and have potential implications towards predicting nanoparticle biocompatibility.     

Direct detection of proteins adsorbed on synthetic materials by matrix-assisted laser desorption ionization-mass spectrometry.

The irreversible accumulation of biological material on synthetic surfaces ("biofouling") adversely affects for instance contact lenses, implantable biomedical devices, biosensors, water purification, transport and storage systems, and marine structures. It is shown here that proteins adsorbed on contact lenses can be detected directly, rapidly, and conveniently, with high sensitivity, by matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers on samples retrieved from clinical usage and in vitro protein adsorption studies, at levels substantially below monolayer coverage. Identification of the detected biological molecules can be done by comparison of the detected mass peaks with known protein molecular masses or with spectra recorded of pure compounds or by separate biochemical assays. The MALDI mass spectra recorded on different contact lenses contain peaks assignable to lysozyme and a number of smaller proteins. Such sensitive characterization of the early stages of biofouling enhances the understanding of protein/materials interactions and assists in designing guided strategies toward control of biological adsorption processes.     

New nanocomposite films for biosensors: layer-by-layer adsorbed films of polyelectrolytes, proteins or DNA

This report describes the construction of ultrathin multicomponent films with an internal structure on the nanometre scale. In the simplest case, the films are built-up by the subsequent adsorption of polyanions and polycations. They can be fabricated on inorganic substrates such as glass, quartz or silicon wafers, or on various organic materials. The polymeric interlayers can incorporate materials with desired electrical or optical properties. The average thickness of the layers can be fine-tuned with Angstrom precision by the addition of suitable salts. They are temperature stable up to at least 200°C and can be laterally structured using conventional photolithographic techniques. The films provide for a well-defined substrate-independent interface for the immobilization of biological macromolecules, such as proteins or DNA, in their active state. The immobilization of streptavidin enables the controlled attachment of any biotinylated molecule with no resulting loss in its biological activity. Area-selective immobilization provides the possibility of built-in quality control for the fabrication of biosensors with separated reference and sample areas on the same substrate.     

Proteome analysis of human serum proteins adsorbed onto different titanium surfaces used in dental implants

Titanium dental implants are commonly used due to their biocompatibility and biochemical properties; blasted acid-etched Ti is used more frequently than smooth Ti surfaces. In this study, physico-chemical characterisation revealed important differences in roughness, chemical composition and hydrophilicity, but no differences were found in cellular in vitro studies (proliferation and mineralization). However, the deposition of proteins onto the implant surface might affect in vivo osseointegration. To test that hypothesis, protein layers formed on discs of both surface type after incubation with human serum were analysed. Using mass spectrometry (LC/MS/MS), 218 proteins were identified, 30 of which were associated with bone metabolism. Interestingly, Apo E, antithrombin and protein C adsorbed mostly onto blasted and acid-etched Ti, whereas the proteins of the complement system (C3) were found predominantly on smooth Ti surfaces. These results suggest that physico-chemical characteristics could be responsible for the differences observed in the adsorbed protein layer.