Dynamic analysis of irreversible adsorption of protein on porous polymer resins as studied by pulse injection method

Irreversible adsorption of bovine serum albumin onto porous polymer resins is examined by a pulse injection method. The experimental results are theoretically analyzed by a model based on the assumption that there exist three kinds of binding sites with different binding rates, which are considered to exist in the vicinity of the outer periphery surface, the inner surface of macropore, and the micropore, or center part, of particles. The three kinds of adsorption rates are also evaluated by a batch method and are nearly equal to the corresponding kinetic parameters by this method. The amount of BSA bound irreversibly to the resins is independent of the protein concentration and the flow rate examined, which suggests that protein molecules penetrate into pores of resins and occupy almost all the effective binding sites in the resins. The total amount of bound BSA shows a pH dependence with a maximum near the isoelectric point of BSA, and the amount of BSA bound at the slowest rate is most largely influenced by pH.     

Effect of Air Plasma Processing on the Adsorption Behaviour of Bovine Serum Albumin on Spin-Coated PMMA Surfaces

This paper reports the adsorption of Bovine Serum Albumin （BSA） onto Dielectric Barrier Discharge （DBD） processed Poly（methyl methacrylate） （PMMA） surfaces by a Quartz Crystal Microbalance with Dissipation monitoring （QCM-D） technique. The purpose is to study the influence of DBD processing on the nature and scale of BSA adsorption on PMMA surface in vitro. It was observed that DBD processing improves the surface wettability of PMMA film, a fact attributable to the changes in surface chemistry and topography. Exposure of the PMMA to Phosphate Buffed Saline （PBS） solution in the QCM-D system resulted in surface adsorption which reaches an equilibrium after about 30 minutes for pristine PMMA, and 90 minutes for processed PMMA surface. Subsequent injection of BSA in PBS indicated that the protein is immediately adsorbed onto the PMMA surface. It was revealed that adsorption behaviour of BSA on pristine PMMA differs from that on processed PMMA surface. A slower adsorption kinetics was observed for pristine PMMA surface, whilst a quick adsorption kinetics for processed PMMA. Moreover, the dissipation shift of protein adsorption suggested that BSA forms a more rigid structure on pristine PMMA surface that on processed surface. These data suggest that changes in wettability and attendant chemical properties and surface texture of the PMMA surface may play a significant role in BSA adsorption process.     

Adsorption kinetics and equilibria of bovine serum albumin on rigid ion-exchange and hydrophobic interaction chromatography matrices in a stirred cell

Rigid adsorbents have advantages over soft gel media for downstream processing of proteins. The adsorption of bovine serum albumin (BSA) has been investigated on a rigid adsorbent based on a wide-pore, hydrophilically coated, silica-gel matrix. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction chromatography) and particle size have been studied on the physical properties of the adsorbent and on the adsorption equilibria and adsorption kinetics. The rates of adsorption of BSA have been measured in a stirred cell and are found to be satisfactorily described by a two-step theoretical model, in which the mass transfer involves a pore diffusion resistance and an extra-particle film resistance. On the anion exchanger, the effective pore diffusivity decreases substantially with increasing protein concentration, approximately halving as the initial concentration rises from 0.7 to 2g/l. In the hydrophobic interaction chromatography medium, the pore diffusivity is less sensitive to protein concentration and is also reduced by a factor of about 4 by aggregation of the protein. Effective pore diffusivities with the "wide-pore" silica adsorbents in anion-exchange form are 36-94 times lower than the diffusivity in free solution and are comparable with the lower of the wide range of values published for soft gels.     

Adsorption and desorption kinetics of bovine serum albumin in ion exchange and hydrophobic interaction chromatography on silica matrices

Large scale chromatographic separation of proteins can be carried out more rapidly on rigid adsorbents than on soft gel media. The kinetics of adsorption of bovine serum albumin (BSA) have been studied on rigid adsorbents based on a wide-pore, hydrophilically-coated silica gel matrix in a packed bed (chromatographic column). Process parameters have been varied comprehensively. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction), particle size and liquid flow velocity have been studied on both the adsorption and desorption processes. The relative influences of the adsorption kinetics and equilibrium isotherm on the shape of the breakthrough curve are found to vary with the process parameters in an interpretable and therefore, predictable manner. Pore diffusion resistance is dominant over the external liquid film resistance in controlling the adsorption kinetics, with Biot numbers in the range 170-2600. A two-step model based on these two resistances simulates the breakthrough curves with only limited quantitative accuracy, but gives good predictions of the effect of changes in process parameters.     

The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA).poly(dT)

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.     

Interaction of Bacteriophage N1 with Cell Walls of Micrococcus lysodeikticus

Bacteriophage N1 does not irreversibly adsorb to cell walls isolated from its host Micrococcus lysodeikticus strain 1 (ML-1). ML-1 walls do bind the virus in a specific but completely reversibly union. Electron microscopic examination of OsO(4)-treated mixtures of phage and walls revealed phage bound to wall fragments by their tail tips, suggesting that reversible phage attachment to walls involves a "tail-first" adsorption of the virus. Treatment of ML-1 walls with fluorodinitrobenzene confers upon the walls the ability to inactivate N1 phage. The relationship between reversible phage attachment to walls and the mechanism of infection by N1 phage is discussed.     

Modification of carbon nanotubes with redox hydrogel: improvement of amperometric sensing sensitivity for redox enzymes

This study demonstrated that redox hydrogel-modified carbon nanotube (CNT) electrodes can be developed as an amperometric sensor that are sensitive, specific and fast and do not require auxiliary enzymes. A redox polymer, poly(vinylimidazole) complexed with Os(4,4'-dimethylbpy)(2)Cl (PVI-dmeOs) was electrodeposited on Ta-supported multi-walled CNTs. The resulted PVI-dmeOs thin film did not change the surface morphology of the CNTs, but turned the CNT surface from hydrophobic to hydrophilic, as studied by scanning electron microscopy (SEM) and static water contact angle measurements. Cyclic voltammetry measurements in a Fe(CN)(6)(3-) solution and electrochemical impedance measurements in an equimolar Fe(CN)(6)(3-/4-) solution demonstrated that the PVI-dmeOs hydrogel thin film was electronic conductive with a resistance of about 15Omega. The PVI-dmeOs/CNT electrodes sensed rapidly, sensitively and specifically to model redox enzymes (glucose oxidase (GOD) and lactate oxidase (LOD)) in amperometric experiments in electrolyte solutions containing the substrates of the measured redox enzymes. Both the CNT substrate and the thin PVI-dmeOs film enhanced the sensing sensitivities. Exploration of the mechanisms suggests that the PVI-dmeOs film may enhance the sensing sensitivities by wiring the enzyme molecules through the redox centers tethered on the mobile redox polymer backbones to the CNT electrodes.     

Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics.

The total internal reflection/fluorescence photobleaching recovery (TIR/FPR) technique (Thompson et al. 1981. Biophys. J. 33:435) is used to study adsorbed bovine serum albumin dynamics at a quartz glass/aqueous buffer interface. Adsorbed fluorescent labeled protein is bleached by a brief flash of the evanescent wave of a focused totally internally reflected laser beam. The rates of adsorption/desorption and surface diffusion determine the subsequent fluorescence recovery. The protein surface concentration is low enough to be proportional to the observed fluorescence and high enough to insure that the observed recovery rates arise mainly from adsorbed rather than bulk protein dynamics. The photobleaching recovery curves for rhodamine-labeled bovine serum albumin reveal both an irreversibly bound state and a multiplicity of reversibly bound states. The relative amount of reversible to irreversible adsorption increases with increasing bulk protein concentration. Since the adsorbed protein concentration appears to be too high to pack into a homogeneous surface monolayer, the wide range of desorption rates possibly results from multiple layers of protein on the surface. Comparison of the fluorescence recovery curves obtained with various focused laser beam widths suggests that some of the reversibly bound bovine serum albumin molecules can surface diffuse. Aside from their relevance to the surface chemistry of blood, these results demonstrate the feasibility of the TIR/FPR technique for measuring molecular dynamics on solid surfaces.     

Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin

A simple adaptation of a commercial spectrofluorometer allows selective excitation of fluorescent biomolecules adsorbed to a solid surface while they are in equilibrium with a bulk solution. As a demonstration of this technique, we have detected a change in the effective singlet-singlet energy transfer in fluorescence-labeled bovine serum albumin (BSA) upon adsorption to a fused silica surface. The technique combines total internal reflection fluorescence excitation of surface-adsorbed BSA with a fluorescence spectroscopic examination of energy transfer between two different fluorophores that are covalently bound to amino groups in each BSA molecule. Two donor--acceptor pairs were used, 4-chloro-7-nitro-2,1,3-benzoxadiazole-rhodamine and dansyl-eosin. For studies of surface-adsorbed BSA, we constructed a device in which the excitation light of a standard fluorescence spectrometer totally internally reflects from a surface at which adsorbed BSA is in equilibrium with the bulk solution. A shallow evanescent wave is created, which excites fluorescence from only those BSA molecules in close proximity to the surface. Spectral examination shows significantly less effective singlet-singlet energy transfer from the donor to the acceptor in surface-adsorbed BSA relative to that in native bulk-dissolved BSA. Under appropriate and reasonable assumptions, the energy transfer change between native and adsorbed states of fluorescent BSA can be interpreted as a conformational change of BSA upon adsorption.     

The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro

Covalent binding of the reactive metabolite of acetaminophen has been investigated in hepatic microsomal preparations from phenobarbital-pretreated mice. Low molecular weight thiols (cysteine and glutathione) were found to inhibit this binding, whereas several other amino acids which were tested did not. Bovine serum albumin (BSA), which contains a single free sulfhydryl group per molecule and which thus represents a macromolecular thiol compound, inhibited covalent binding of the reactive acetaminophen metabolite to microsomal protein in a concentration-dependent manner. The acetaminophen metabolite also became irreversibly bound to BSA in these experiments, although this binding was reduced by approx. 47% when the thiol function of BSA was selectively blocked prior to incubation. Covalent binding of the acetaminophen metabolite to bovine alpha s1-casein, a soluble protein which does not contain any cysteine residues, was found to occur to an extent of 37% of that which became bound to native BSA. These results were taken to indicate that protein thiol groups are major sites of covalent binding of the reactive metabolite of acetaminophen in vitro. The covalent binding characteristics of synthetic N-acetyl-p-benzoquinoneimine (NAPQI), the putative electrophilic intermediate produced during oxidative metabolism of acetaminophen, paralleled closely those of the reactive species generated metabolically. These findings support the contention that NAPQI is indeed the reactive arylating metabolite of acetaminophen which binds irreversibly to protein.     

Irreversible Binding of [3H]Flunitrazepam to Different Proteins in Various Brain Regions

Irreversible photolabeling by [3H]flunitrazepam of four proteins with apparent molecular weights 51,000 (P51), 53,000 (P53), 55,000 (P55), and 59,000 (P59) was investigated in various rat brain regions by SDS-polyacrylamide gel electrophoresis, fluorography, and quantitative determination of radioactivity bound to proteins. On maximal labeling of these proteins, only 15-25% of [3H]flunitrazepam reversibly bound to membranes becomes irreversibly attached to proteins. Results presented indicate that for every [3H]flunitrazepam molecule irreversibly bound to membranes, three molecules dissociate from reversible benzodiazepine binding sites. This seems to indicate that these proteins are either closely associated or identical with reversible benzodiazepine binding sites, and supports the hypothesis that four benzodiazepine binding sites are associated with one benzodiazepine receptor. When irreversible labeling profiles of proteins P51, P53, P55, and P59 were compared in different brain regions, it was found that labeling of individual proteins varied independently, supporting previous evidence that these proteins are associated with distinct benzodiazepine receptors.     

BSA Adsorption on Porous Scaffolds Prepared from BioPEGylated Poly(3-Hydroxybutyrate)

Porous scaffolds for tissue engineering have been prepared from poly(3-hydroxybutyrate) (PHB) and a copolymer of poly(3-hydroxybutyrate) and polyethylene glycol (PHB-PEG) produced by bioPEGylation. The morphology of the scaffolds and their capacity for adsorption of the model protein bovine serum albumin (BSA) have been studied. Scaffolds produced from bioPEGylated PHB adsorbed more BSA, whereas the share of protein irreversibly adsorbed on these scaffolds was significantly lower (33%) than in the case of PHB homopolymer-based scaffolds (47%). The effect of protein adsorption on scaffold biocompatibility in vitro was tested in an experiment that involved the cultivation of fibroblasts (line COS-1) on the scaffolds. PHB-PEG scaffolds had a higher capacity for supporting cell growth than PHB-based scaffolds. Thus, the bioPEGylated PHB-based polymer scaffolds developed in the present study have considerable potential for use in soft tissue engineering.     

The influence of radioiodination on the adsorption of IgG and serum albumin to polystyrene

The adsorption of radioiodinated rabbit IgG and bovine serum albumin (BSA) to polystyrene tubes was investigated. Adsorption isotherms where the proportion of the protein bound was relatively constant over a range of intermediate protein concentrations, and where the proportion bound was protein dependent, were obtained. To investigate the effects of radioiodination, proteins labeled to give a wide range of substitution ratios (0.03 to 3.7 125I/protein molecule) were employed. While labeling did not appear to affect BSA adsorption, the kinetics of IgG binding were altered in a number of ways. The proportion bound in the concentration independent region was decreased even at substitution ratios less than or equal to 0.2. In addition, while all preparations of iodinated BSA, and IgG preparations with less than or equal to 1.6 125I/IgG, gave bimodal adsorption isotherms, with more heavily labeled IgG (greater than or equal to 2.5 125I/IgG) the apparent high affinity binding to the plastic surface was abolished. These results indicate that radioiodination substantially alters the kinetics of the binding of IgG to polystyrene. In addition, the results obtained are discussed with respect to previous relevant and often apparently contradictory findings.     

Competitive protein adsorption on polysaccharide and hyaluronate modified surfaces

Adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) was measured on six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and Streptococcus zooepidemicus. Film thickness and surface morphology depended on the HA molecular weight and concentration. BSA coverage was enhanced on surfaces in competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of HA utilized. With changing bulk protein concentration from 20 to 40 μg ml(-1) for each species, Fg coverage on silicon increased by 4x, whereas both BSA and Fg adsorption on dextran and HA were far less dependent on protein bulk concentration.     

A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins

Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles occurs by a nonendocytotic pathway that requires no specific apolipoprotein and results in the net delivery of cholesteryl esters to cells. Here we examine a reversibly cell-associated pool of cholesteryl ester tracer and its relationship to selective uptake. A fraction of cholesteryl ester tracer selectively taken up from HDL by rat primary or mouse Y1-BS1 adrenocortical cells was chased from the cells by subsequent incubation with unlabeled HDL. This pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL particles bound to the cell surface. In response to various metabolic effectors, cholesteryl ester tracer in this reversibly cell-associated pool of Y1-BS1 cells correlated linearly with irreversible selective uptake. Both reversibly and irreversibly cell-associated pools of cholesteryl ester tracer displayed similar saturation kinetics for uptake from HDL, and both pools correlated inversely with cell-free cholesterol levels. Cholesteryl ester tracer in the reversible pool was shown to serve as a precursor for irreversible selective uptake. A pool with properties similar to the reversibly cell-associated pool was identified in plasma membrane fractions; enough tracer was incorporated into this pool to account for the reversibly cell-associated pool of intact cells. The data suggest that a pool of cholesteryl esters in the plasma membrane is involved in selective uptake at a step prior to irreversible internalization.     

An analysis of the interaction of protein with lipid monolayers at the air/water interface

1. Measurements have been made of the interaction of cytochrome c, bovine serum albumin and synthetic oxytocin with low-pressure (2dyn/cm) monolayers of stearic acid, phosphatidylcholine and phosphatidylethanolamine. 2. [(14)C]Carboxymethylation of the cytochrome c and albumin followed by surface-radioactivity determinations have shown that only a proportion of the protein added to the subphase is bound to the monolayers and that initially the degree of binding is dependent on the protein concentration. The binding is irreversible in the sense that the adsorbed protein cannot be removed by transferring the film containing the interacted protein to a fresh subphase containing no protein. 3. Three successive types of interaction can usually be recognized. (a) Initially, whole molecules of protein penetrate the lipid film and occupy the same area as those of the protein spread at the air/water interface. (b) Above certain film pressures a part of each protein molecule, probably hydrophobic side chains, penetrates the film. The change in surface pressure per unit of bound protein is much smaller than in (a). (c) At higher film pressures, adsorption without penetration occurs. With cytochrome c this is initially dependent on a favourable electrostatic interaction.     

Heparin conjugated polylactide as a blood compatible material

A heparin-conjugated biodegradable polymer (PLA-heparin) by the direct coupling of heparin to polylactide (PLA) was synthesized and characterized. The surface exposed heparin content associated PLA-heparin was measured to be 0.067 microg/cm2. PLA-heparin coated surface has shown higher hydrophilicity rather than control PLA surface. The clotting time of PLA-heparin conjugate measured by activated partial thromboplastin time (APTT) was significantly prolonged as compared to PLA. The bioactivity of bound heparin measured by APTT corresponds to 17.4% of free heparin. It has been also demonstrated that the conjugation of heparin suppresses the protein adsorption as well as the platelet adhesion. These results indicate that the unique property of bound heparin has an inhibiting influence on the coagulation, plasma protein adsorption, and subsequent platelet adhesion systems. This novel PLA-heparin conjugate could be applied as blood/tissue compatible biodegradable materials for implantable medical devices and tissue engineering.     

Electrostatic-assembly-driven formation of supramolecular rhombus microparticles and their application for fluorescent nucleic acid detection

In this paper, we report on the large-scale formation of supramolecular rhombus microparticles (SRMs) driven by electrostatic assembly, carried out by direct mixing of an aqueous HAuCl(4) solution and an ethanol solution of 4,4'-bipyridine at room temperature. We further demonstrate their use as an effective fluorescent sensing platform for nucleic acid detection with a high selectivity down to single-base mismatch. The general concept used in this approach is based on adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by SRM, which is accompanied by substantial fluorescence quenching. In the following assay, specific hybridization with its target to form double-stranded DNA (dsDNA) results in desorption of ssDNA from SRM surface and subsequent fluorescence recovery.     

Zinc-decorated silica-coated magnetic nanoparticles for protein binding and controlled release

The aim of this study was to be able to reversibly bind histidine-rich proteins to the surface of maghemite magnetic nanoparticles via coordinative bonding using Zn ions as the anchoring points. We showed that in order to adsorb Zn ions on the maghemite, the surface of the latter needs to be modified. As silica is known to strongly adsorb zinc ions, we chose to modify the maghemite nanoparticles with a nanometre-thick silica layer. This layer appeared to be thin enough for the maghemite nanoparticles to preserve their superparamagnetic nature. As a model the histidine-rich protein bovine serum albumin (BSA) was used. The release of the BSA bound to Zn-decorated silica-coated maghemite nanoparticles was analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrated that the bonding of the BSA to such modified magnetic nanoparticles is highly reversible and can be controlled by an appropriate change of the external conditions, such as a pH decrease or the presence/supply of other chelating compounds.     

The interaction of cytochrome c with monolayers of phosphatidylethanolamine

1. The interaction between [(14)C]carboxymethylated cytochrome c and monolayers of egg phosphatidylethanolamine at the air/water interface has been investigated by measurements of surface radioactivity, pressure and potential. 2. On adding (14)C-labelled cytochrome c to the subphase under monolayers with a surface pressure below 24dynes/cm. there was an initial surface pressure increment as the protein penetrated, followed by an adsorption that could be detected only by a continued increase in the surface radioactivity. 3. Above film pressures of 24dynes/cm. only adsorption was observed, i.e. an increment in surface radioactivity with none in surface pressure. 4. The changes in surface parameters with penetration of cytochrome c added to the subphase were indirectly proportional to the initial pressure of the monolayer. With hydrogenated phosphatidylethanolamine the constant of proportionality was increased but penetration again ceased at 24dynes/cm. 5. On compressing a phosphatidylethanolamine film containing penetrated cytochrome c to 40dynes/cm. only a proportion of the protein was ejected on a subphase of 10mm-sodium chloride, whereas on a subphase of m-sodium chloride nearly all the protein was lost. 6. With both penetration and adsorption only a small proportion of the added cytochrome c interacted with the phospholipid films, and initially the amount bound was proportional to the added protein concentration. There was no evidence of a stoicheiometric relationship between the protein and phospholipid or the build-up of multilayers. The bonded protein was not released by removing cytochrome c from the subphase. 7. The addition of m-sodium chloride to the subphase delays the rate of protein penetration into low-pressure films, but the final surface-pressure increment is not appreciably decreased. In contrast, m-sodium chloride almost completely stops adsorption on to films at all pressures. 8. When sodium chloride is added to the subphase below cytochrome c adsorbed to monolayers at high pressures, so that the final concentration is 1m, only a proportion of the protein is desorbed and this decreases as the time of the interaction increases. This indicates that adsorption is initially electrostatic, followed by the formation of non-ionic bonds. 9. Alteration of the subphase pH under a high-pressure film leads to a steady increase in adsorption from pH3 to 8.5 followed by a rapid fall to zero adsorption at pH11. 10. The penetration into phospholipid monolayers at 10dynes/cm. shows a rate that is consistent with the relative electrostatic status of the two components of the interaction as the subphase pH is varied between 3 and 10.5. The final equilibrium penetration shows a pronounced peak in the increments of surface pressure at pH9.0 although a similar peak is not observed in the surface radioactivity. This indicates that more residues of the protein are penetrating into the film at about this pH. 11. Determinations were made of the electrophoretic mobilities of phosphatidylethanolamine particles both alone and after interaction with cytochrome c. 12. The electrophoretic mobilities of cytochrome c adsorbed on lipid particles showed an isoelectric point below that of cytochrome c. This and the observations on the monolayers suggest that, with cytochrome c, protein-protein interactions are weak compared with other proteins.