A NOVEL METAL CHELATE AFFINITY ADSORBENT FOR PROTEIN UPTAKE

In this article,a spherical chitosan gel crosslinked by epichlorohydrin was prepared.It was then loaded with copper ions to produce a metal chelate affinity adsorbent for protein.The uptake of bovine serum albumin(BSA)by the affinity adsorbent was investigated.and the adsorption capacity for BSA as high as 40mg/g-wet beads was observed.The adsorption equilibrium data was well correlated by the Langmuir equation.The adsorption was considerably affected by pH.In additio.The amount of BSA adsorbed onto the beads decreased with the increasing of aqueous phase ionic strength,so adsorbed BAS can be desorbed by adjusting pH orionic strength of the solution.     

Adsorption of monoclonal IgG on polystyrene microspheres

In this paper the adsorption of a monoclonal antibody IgG-1 isotype against HBsAg onto positively and negatively charged polystyrene beads has been studied. To determine the role played by electrostatic forces in the adsorption process different pH values were used. It was confirmed that the affinity of adsorption isotherms depends on the electrostatic interaction between protein and polymer surface. The maximum adsorption amount is located around the i.e.p. of the dissolved protein, and decreases markedly as pH moves away. Thus, the major driving force for adsorption of monoclonal antibodies on polystyrene beads comes from the hydrophobic interaction between the antibody molecules and the adsorbent surface. Desorption of preadsorbed IgG molecules by increasing ionic strength has shown that the positively charged polystyrene is also more hydrophobic in character than the negatively charged one. Finally, electrokinetic experiments have determined that the electric double layer (e.d.l.) of monoclonal antibody changes as the consequence of adsorbing on charged polymer surfaces.     

Adsorption behavior of BSA in microfiltration with porous glass membrane

The fouling mechanism during dead-end microfiltration of bovine serum albumin (BSA) with porous glass membrane was investigated from the point of BSA adsorption onto the pore surface of membrane under the condition of pH 5.0 and ionic strength 0.01. The location of BSA retention was confirmed by comparing the filtration performance between dead-end mode and cross-flow mode. During the dead-end microfiltration BSA was retained only by the adsorption on the pore surface. The adsorption was irreversible and of multilayer type, which consists of the adsorption on clean pore surface, i.e. the primary adsorption, and that on preadsorbed pore surface, i.e. the secondary one. The adsorption isotherm was high affinity type. The adsorption rate was proportional to the feed rate of BSA, and the proportional coefficient was dependent on the adsorption process. The flux decline was correlated quantitatively with the amount of adsorbed BSA from the pore radius narrowing model by adsorption.     

Characterization of a planar poly(acrylic acid) brush as a materials coating for controlled protein immobilization

The adsorption of two different proteins at a planar poly(acrylic acid) (PAA) brush was studied as a function of the ionic strength of the protein solutions applying total internal reflection fluorescence (TIRF) spectroscopy. Planar PAA brushes were prepared with a grafting density of 0.11 nm(-2) and were characterized using X-ray reflectometry. Hen egg-white lysozyme and bovine serum albumin (BSA) were used as model proteins, which have a net positive and negative charge at neutral pH-values, respectively. It has been found that both proteins adsorb strongly at a planar PAA brush at low ionic strength. Whereas lysozyme interacts with a PAA brush under electrostatic attraction at neutral pH-values, BSA binds under electrostatic repulsion at pH > 5. Even at pH = 8, significant amounts of BSA are adsorbed to a planar PAA brush. In addition, the reversibility of BSA adsorption has been characterized. Dilution of a BSA solution leads to an almost complete desorption of BSA from a PAA brush at short contact times. When the ionic strength of the protein solutions is increased to about 100-200 mM, a planar PAA brush appears largely protein-resistant, regardless of the protein net charge. The results of this study indicate that the salt-dependent protein affinity of a PAA brush represents a unique effect that must be explained by a novel protein-binding mechanism. On the basis of a recent model, it is suggested that a release of counterions is the most probable driving force for protein adsorption at a PAA brush. In a general view, this study characterizes a planar PAA brush as a new materials coating for the controlled immobilization of proteins whose use in biotechnological applications appears to be rewarding.     

Magnetic dye affinity beads for the adsorption of beta-casein

Casein is well known as a good protein emulsifier and beta-casein is the major component of casein and commercial sodium caseinate. Dye affinity adsorption is increasingly used for protein separation. beta-Casein adsorption onto Reactive Red 120 attached magnetic poly(2-hydroxyethyl methacrylate) (m-PHEMA) beads was investigated in this work. m-PHEMA beads (80-120 microm in diameter) were produced by dispersion polymerization. The dichlorotriazine dye Reactive Red 120 was attached covalently as a ligand. The dye attached beads, having a swelling ratio of 55% (w/w) and carrying different amounts of Reactive Red 120 (9.2 micromol . g(-1)-39.8 micromol . g(-1)), were used in beta-casein adsorption studies. The effects of the initial concentration, pH, ionic strength and temperature on the adsorption efficiency of dye attached beads were studied in a batch reactor. The non-specific adsorption on the m-PHEMA beads was 1.4 mg . g(-1). Reactive Red 120 attachment significantly increased the beta-casein adsorption up to 37.3 mg . g(-1). More than 95.4% of the adsorbed beta-casein was desorbed in 1 h in a desorption medium containing 1.0 M KSCN at pH 8.0. We concluded that Reactive Red 120 attached m-PHEMA beads can be applied for beta-casein adsorption without significant losses in the adsorption capacities.     

Removal of endotoxin from culture supernatant of Bordetella pertussis with aminated poly(gamma-methyl L-glutamate) spherical beads.

Attempts were made to prepare adsorbents having a high affinity for endotoxin in the culture supernatant of Bordetella pertussis. When poly(gamma-methyl L-glutamate) (PMLG) was used as a matrix and amino groups as the ligand, the highest affinity for endotoxin was attained even at a high ionic strength (mu = 0.2-0.4). PMLG beads containing amino groups of about 3.2 meq/g selectively removed endotoxin from the culture supernatant of B. pertussis without affecting the protective antigens. It was demonstrated that 1 ml of the wet adsorbent adsorbed 4.5 mg of endotoxin. The beads of PMLG derivatives, therefore, are considered to be a useful adsorbent for the removal of endotoxin from the pertussis vaccine, affecting neither filamentous hemagglutinin nor pertussis toxin.     

壳聚糖亲和磁性毫微粒的制备及其对蛋白质的吸附性能研究

以壳聚糖为包裹材料包埋自制的磁流体 ,制备了具有核 壳结构的磁性毫微粒 ,并偶联色素配基CibacronBlue 3GA(偶联量 1 4 .5μmol/mL)得到了一种新型亲和磁性毫微粒 .结果表明 ,所得亲和磁性微球具有较窄的粒径分布、形状规整 .以牛血清白蛋白 (BSA)和溶菌酶 (Lys)为目标蛋白 ,考察了该亲和磁性毫微粒的吸附性能 ,发现其对BSA和Lys的吸附量分别为 4和 2 8mg/g,吸附行为满足Langmuir吸附等温式 ,且对时间依赖性小而对溶液离子强度敏感 .     

Adsorption of Bovine Serum Albumin onto Polystyrene Latex Particles Bearing Saccharidic Moieties

The adsorption of bovine serum albumin (BSA) onto polystyrene latexes bearing various amounts of sugar moieties has been investigated as a function of pH and ionic strength and the results were compared to those for bare polystyrene latexes having negative surface charges. The functionalized latexes were produced by seeded copolymerization of (0.3 μm) liposaccharidic monomer onto polystyrene particles obtained by soap-free emulsion polymerization of styrene using potassium persulfate as initiator. At first, the electrophoretic mobility behavior of the various latexes was examined as a function of pH: a significant decrease was observed in the case of saccharide-containing latex particles compared to the bare particles. The adsorption of BSA onto these latexes exhibited a reduced amount of adsorbed BSA for those latex particles bearing saccharide groups. This adsorbed amount depends on the yield of saccharidic monomer incorporated onto the surfaces of the latex particles.     

聚四氟乙烯纤维的改性及其对胆红素的吸附

以聚四氟乙烯(PTFE)纤维为基质, 以甲基丙烯酸缩水甘油酯(GMA)为单体, 通过辐照接枝聚合制备了PTFE-g-GMA纤维, 用聚乙烯亚胺(PEI)与PTFE-g-GMA纤维进行开环反应制得新型吸附剂PTFE-g-GMA-PEI. 考察了PTFE-g-GMA-PEI吸附剂对溶液中胆红素的吸附动力学、吸附量及pH、离子强度及温度等因素对胆红素吸附的影响. 实验结果表明, 该吸附剂对胆红素有较高的吸附容量及良好的选择吸附性能, 其吸附行为遵循Langmuir吸附模式.     

Removal of Toxic As (V) Ions by Adsorption onto Alginate and Carboxymethyl Cellulose Beads

The removal of arsenic ions from dilute aqueous solutions using biopolymeric beads of crosslinked sodium alginate and carboxymethyl cellulose (CMC) as the adsorbent is reported in this paper. The biopolymeric alginate and carboxymethyl cellulose beads were prepared and characterized by FTIR spectra. On the surfaces of the prepared beads were performed static and dynamic adsorption studies of arsenic ions at fixed pH and ionic strength of the aqueous metal ion solutions. The adsorption data were applied to Langmuir and Freundlich isotherm equations and various static parameters were calculated. The dynamic nature of adsorption was quantified in terms of several kinetic constants such as rate constant for adsorption (k₁) and Lagergreen rate constant (K_ad). The influence of various experimental parameters such as solid to liquid ratio, pH, temperature, presence of salts and chemical composition of biopolymeric beads were investigated on the adsorption of arsenic ions.     

Preparation of Functional Fluorescent Microspheres Used for HTS and Their Interaction with Biomolecules

There is considerable interest in protein adsorption onto microspheres because of its importance in a wide range of biomedical applications, such as artificial tissues and organs, drug delivery systems, biosensors, solid-phase immunoassays, immunomagnetic cell separation and immobilized enzymes or catalyst. It has been well known that the interaction between proteins and microspheres plays important roles in this process. Major interaction involved in the adsorption can be classified as electrostatic, hydrophobic and hydrogen-bonding. Indeed, adsorption of proteins onto microspheres is a complex process and often can involve many dynamic steps, from the initial attachment of the protein on the surface of microspheres to the equilibrium. Also the conformation of proteins probably occurs to a certain degree of deformation or structural change due to the large area of contact. Recently, much interest has been shown in sulfonated microspheres, since sulfonate-group itself is one of components in bio-bodies, as well as is sensitive to the change of pH or ionic strength. Indeed, so far, scanty investigations have been performed in the full range. Also few researches have involved the data on adsorption rate and the maximum amount of protein adsorbed, or the reversibility of the process and conformational change of protein adsorbed as well.In present study, BSA (bovine serum albumin) was chosen as the model protein and sulfonated PMMA [poly(methyl methacrylate)] microspheres as the matrix to investigate the adsorption process.The purpose is to show some information especially the intrinsic information involved by the adsorption process Adsorption of BSA onto sulfonated microspheres (MS) has been investigated as a function of time, protein concentration and pH. The adsorption appears to be a reversible process and the presence of sulfonate groups can play important roles in the adsorption process, so as to increase the amount of protein adsorbed and influences the interaction of BSA molecules. Fig. 1 also shows that the reciprocation between unadsorbed and adsorbed BSA or rearrangement of adsorbed BSA molecules does not produce visible change in the properties of the adsorbed protein. Close to the isoelectric point of BSA (pI 4.7), the amount of protein adsorbed exhibits a maximum. A higher or lower pH results in the significant decrease of the adsorption amount. This is related to the dependence of BSA conformations at different pH conditions.     

Diffusion into and adsorption of polyethylenimine on porous silica gel

The polyethyleneimine (PEI)–water–silica gel absorption system was used as a model system to investigate the relationship between diffusion into the porous structure, adsorption rate, and molecular weight of the polymer. Three silica gels, Porasil A, B, and and C having a range of characteristic porosity were used as adsorbents. Adsorption of PEI on Porasil C, which has the majority of its pores much larger than the dimensions of the adsorbate molecule, increased initially with increased molecular weight but became nearly constant at higher molecular weight. Little increase in adsorption occurred for this silica gel with increased ionic strength or with increased pH between 9.5 and 10.8. In contrast, adsorption increased sharply with increased ionic strength and for the same pH range on Porasil A. Molecular weight dependence was reversed. Adsorption decreased with increased molecular weight on Porasil A. In this case, the molecular size of PEI investigated was the same as the majority of pore apertures in the adsorbent. Solution environments (i.e., pH and ionic strength) that decrease the size of the PEI molecule and its affinity for the anionic silica gel surface, thus enabling it to more readily diffuse into the smaller porous regions of the adsorbent, are the apparent causes of the very large adsorption increase. Electrostatic repulsion between PEI molecules do not appear greatly to affect adsorption. Similar adsorption behavior has been reported in the literature for the PEI–cellulosic fiber adsorption system. Maximum adsorption on Porasil A occurred at pH 10.8, the same maximum generally reported for adsorption of PEI on cellulosic fibers. In this case, the silica gel (Porasil A) was found to have a pore size distribution and specific surface area of the same magnitude as cellulosic fibers prepared in the expanded state.     

Properties of protein adsorption onto pore surface during microfiltration: Effects of solution environment and membrane hydrophobicity

Properties of bovine serum albumin (BSA) adsorption onto pore surface during the filtration of BSA containing solution with the Sirasu porous glass membrane with a pore size of 0.1 μm were studied. The effects of pH, ionic strength, and surface modification on the flux decline and breakthrough curves were observed. The adsorption properties of BSA were estimated quantitatively by using the internal fouling model, which relates the filtration performance to the adsorption interaction, the adsorption capacity, and the thickness of the adsorption layer. The electrostatic interaction between BSA and pore surface was estimated by the streaming potential measurement. The BSA adsorption involved a rapid adsorption in the early stage of filtration followed by a slow multilayer adsorption that dominates the long-term filtration performance. The electrostatic repulsive force reduced the overall adsorption interaction but the electrostatic attractive force did not affect the adsorption interaction. The effect of ionic strength on the BSA adsorption could be explained in terms of the shift of the IEP of BSA toward lower pH with the increase in ionic strength. The hydrophobicity of membrane did not affect the adsorption properties except for the adsorption interaction in the early stage of the filtration.     

Selective uptake of different proteins by annealed and quenched cationic spherical polyelectrolyte brushes

The selective uptake of bovine serum albumin (BSA) and β-glucosidase (β-G) by annealed and quenched cationic spherical polyelectrolyte brushes (SPB) was systematically studied by combining turbidimetric titration, dynamic light scattering and small angle X-ray scattering (SAXS). These two kinds of SPB consist of a same polystyrene core and a dense shell of poly (2-aminoethyl methacrylate hydrochloride) (PAEMH) and poly [2-(methacryloyloxy) ethyl] trimethylammonium chloride (PMAETA), respectively. Results reveal that the adsorption/desorption of proteins on SPB can be easily controlled by changing external conditions (pH and ionic strength). For a particular annealed or quenched SPB, there is a significant difference of the interaction pH regions between the brush and the two proteins, and this difference can be tuned by ionic strength. At low ionic strength, quenched brushes were more suitable for selective adsorption of BSA and β-G, while annealed brushes performed better at high ionic strength. SAXS analysis demonstrated that volume exclusion effect played a remarkable role in protein uptake by both SPB, and larger proteins were more likely to be adsorbed on the outer layer of the brush. The unique core-shell structure and controllable chain types make SPB an excellent candidate in selective adsorption/separation of proteins of different sizes.     

Concanavalin a immobilized affinity adsorbents for reversible use in yeast invertase adsorption

Concanavalin A (Con A) immobilized poly(2-hydroxyethyl methacrylate) (PHEMA) beads were investigated for specific adsorption of yeast invertase from aqueous solutions. PHEMA beads were prepared by a suspension polymerization technique with an average size of 150-200 microm, and activated by epichlorohydrin. Con A was then immobilized by covalent binding onto these beads. The maximum Con A immobilization was found to be 10 mg/g. The invertase-loading capability of the PHEMA/Con A beads was 107 mg/g. The maximum invertase adsorption capacity on the PHEMA/Con A adsorbents was observed at pH 5.0. The values of the Michaelis constant K(m) of invertase were significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V(max) was smaller for the adsorbed invertase. Adsorption improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments. Storage stability of adsorbed invertase.     

静电力驱动蛋白质在疏水蛋白表面的吸附

疏水蛋白是丝状真菌产生的一种外泌蛋白质, 它们可以在不同表面形成双亲性蛋白膜. 疏水蛋白也是一种优良的蛋白质固定化基质, 然而蛋白质在疏水蛋白表面吸附的驱动机制却是未知的. 本文系统研究了不同pH和离子浓度下蛋白质在疏水蛋白表面的吸附. 首先, 用石英晶体微天平技术研究了不同pH和离子浓度下, Ⅰ型疏水蛋白HGFI和Ⅱ型疏水蛋白HFBI在聚苯乙烯表面的吸附. 结果发现, pH和离子强度对HGFI在聚苯乙烯表面的吸附影响较大, 对HFBI的吸附影响与HGFI相比则较小; HGFI在聚苯乙烯表面主要形成的是弹性膜, 而HFBI在聚苯乙烯表面主要形成的是刚性膜. 随后又研究了不同pH和离子浓度下牛血清白蛋白(BSA)和亲和素(Avidin)在HGFI和HFB上吸附, 结果表明, pH和离子强度对BSA和Avidin在HGFI和HFB上吸附有显著影响, 说明BSA和Avidin在两种疏水蛋白上吸附的主要驱动力为静电力. 本文研究结果为实现疏水蛋白表面可控地固定蛋白质提供了理论指导.     

Selective Desorption of Intercalated Bovine Serum Albumin and Lysozyme by Organically Modified Montmorillonite

The organically modified montmorillonite (M‐Mt) was applied as an adsorbent for the purification of bovine serum albumin (BSA). In order to differentiate the selectivity and perform the purification of BSA, two kinds of proteins, BSA and lysozyme (LYZ) were mixed together and prepared at different pH, which could change the electrical charges on the surfaces of the proteins. BSA and LYZ can be adsorbed at the lower pH into the organically modified montmorillonite, which could be confirmed by powder X‐ray diffraction (XRD) in the d‐value increased after the adsorption of proteins. However, there is only BSA desorption was observed, approved by the method of Sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE), from this adsorbed protein mixture when the pH of the solution was adjusted and optimized. These results indicate that there is electrostatic interaction between a suitably modified montmorillonite and proteins BSA and LYZ to perform the selective desorption from BSA in the mixture of these proteins.     

辛巴蓝F-3GA修饰的啤酒废酵母菌亲和吸附溶菌酶

利用j嗪染料辛巴蓝F-3GA修饰经戊二醛交联的啤酒废酵母菌,得到一种新型染料亲和吸附剂.辛巴蓝F-3GA的固载量为161.1 mg/g.以溶菌酶为研究对象,考察吸附时间、酶初始浓度、pH值、离子强度等因素对吸附率的影响.结果表明:当pH=7.0时,其对溶菌酶有较高的吸附量(229.1 mg/g),吸附性能明显优于未接枝...     

Structural Conformation of Bovine Serum Albumin Layers at the Air-Water Interface Studied by Neutron Reflection

The adsorption of bovine serum albumin (BSA) at the air-water interface has been studied by specular neutron reflection. The variation of the adsorbed amount and the total thickness of the BSA layer with respect to bulk BSA concentration was determined at pH 5, close to its isoelectric point (IP). While the surface excess showed a steady increase with bulk concentration the thickness of the protein layer was found to be close to the short axial length of 40 ? of the globular solution structure of BSA at concentrations below 0.1 g dm-3, suggesting that BSA molecules adsorb with their long axes parallel to the surface of water. At 1 g dm-3 the adsorbed layer can be modeled as an upper layer of 40 ? with a volume fraction of 0.4 and a sublayer of 30 ? underneath the top main layer with a volume fraction of 0.12. The results suggest that, although there is some structural deformation accompanying adsorption, there is no denaturation. The extent of immersion of the BSA in water was determined by performing the measurements in D2O and in a mixture of H2O and D2O whose contrast matches that of BSA. The signal is then only from the part of the layer out of water. At pH 5 this layer was about 10 +/- 5 ? at a bulk concentration of 5 x 10(-4) g dm-3 and decreased to 5 +/- 3 ? at 1 g dm-3. The fraction of the BSA layer immersed in water therefore varies from about 70 to over 90%. The effect of pH on the adsorption was examined at two BSA concentrations. While pH had little effect on the adsorption at a low BSA concentration of 5 x 10(-3) g dm-3, both surface excess and layer thickness showed pronounced peaks at pH 5 at the higher concentration of 1 g dm-3. The increased adsorption at pH 5 is attributed to the reduced lateral electrostatic repulsion around the IP. This adsorption pattern became less pronounced when the total ionic strength was increased from 0.02 to 1 M, indicating that the electrolyte screens the electrostatic repulsions within the adsorbed layer. Copyright 1999 Academic Press.