蛋白芯片载体表面修饰对探针固定影响的研究

采用3-氨基丙基-三甲氧基硅烷((3-aminopropyl)trimethoxysilane,APTES)、戊二醛(glutaraldehyde,GA)、多聚-L-赖氨酸(poly-L-lysine,PLL)修饰芯片载体表面,对3种不同修饰方法制备的蛋白质芯片进行对比研究。将Cy3标记羊抗鼠IgG固定在修饰后片基上,选择蛋白探针的固定率作为检测指标;将小鼠IgG作为探针固定在芯片上,靶蛋白为Cy3标记羊抗鼠IgG,通过生物芯片扫描仪检测反应后荧光强度,选择蛋白探针的反应性作为检测指标,探讨制备蛋白质芯片较佳的表面修饰方法。结果显示,戊二醛修饰玻片对蛋白固定较好,有较高的反应活性,检测限较宽,但背景噪声较高。     

An Equilibrium Model for the Combined Effect of Macromolecular Crowding and Surface Adsorption on the Formation of Linear Protein Fibrils

The formation of linear protein fibrils has previously been shown to be enhanced by volume exclusion or crowding in the presence of a high concentration of chemically inert protein or polymer, and by adsorption to membrane surfaces. An equilibrium mesoscopic model for the combined effect of both crowding and adsorption upon the fibrillation of a dilute tracer protein is presented. The model exhibits behavior that differs qualitatively from that observed in the presence of crowding or adsorption alone. The model predicts that in a crowded solution, at critical values of the volume fraction of crowder or intrinsic energy of the tracer-wall interaction, the tracer protein will undergo an extremely cooperative transition—approaching a step function—from existence as a slightly self-associated species in solution to existence as a highly self-associated and completely adsorbed species. Criteria for a valid experimental test of these predictions are presented.     

The Adsorption of Whey Proteins on the Surface of Emulsified Fat

Whey proteins adsorbed on fat globule surfaces during emulsification with coconut oil at pH 3 ~ 9 were examined. The amount of proteins adsorbed on the fat surface was dependent on the pH during emulsification. At any pH examined here, however, tightly-adsorbed proteins which were not extracted from the fat surface with urea or guanidine-HCl were 2 ~ 3 mg/m². Marked selectivities in the adsorption of individual whey proteins were observed at any pH. No correlation between the adsorbabilities and the surface hydrophobicities of individual whey proteins was observed. Whey proteins adsorbed on the emulsified fat were much more easily digested with proteases compared to the native whey proteins, indicating that conformational changes of whey proteins occurred at the fat surface. The results suggested that conformational properties, such as flexibility of the structure, of whey proteins are important in the adsorption and possibly affect their emulsifying ability.     

Effect of Yeast Cell Morphology,Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.     

Specific Surface Area Effect on Adsorption of Chlorpyrifos and TCP by Soils and Modelling

The adsorption of chlorpyrifos and TCP (3,5,6, trichloro-2-pyridinol) was determined in four soils (Mollisol, Inceptisol, Entisol, Alfisol) having different specific surface areas (19–84 m² g^?1) but rather similar organic matter content (2.4–3.5%). Adsorption isotherms were derived from batch equilibration experiments at 25°C. After liquid-liquid extraction, the chlorpyrifos and TCP concentrations in the solution phase were determined by gas chromatography with an electron capture detector. Adsorption coefficients were calculated using the Freundlich adsorption equation. High K_F coefficients for chlorpyrifos (15.78) and TCP (6.54) were determined for the Entisol soil, while low K_F coefficients for chlorpyrifos (5.32) and TCP (3.93) were observed in the Alfisol soil. In all four soils, adsorption of chlorpyrifos was higher than that of TCP. A surface complexation model, the constant capacitance model, was well able to fit the adsorption isotherms of both chlorpyrifos and TCP on all four soils. The results showed that specific surface area affected adsorption of both chlorpyrifos and TCP. Among the soil properties, specific surface area could be a better indicator than organic matter content alone for adsorption of chlorpyrifos and TCP by soils that contained low organic matter.     

Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins.     

大孔吸附树脂对乳清分离蛋白酶解物的吸附特性研究

研究了大孔吸附树脂对乳清分离蛋白（WPI）酶解液的吸附特性。比较了6种大孔吸附树脂对WPI酶解物的静态吸附率与解吸附率。结果表明,DA201-C大孔吸附树脂最适合WPI酶解物的分离,其对WPI酶解液的动态吸附条件为：上样液浓度：10mg／mL;洗脱剂：75％乙醇溶液;洗脱剂流速：1BV／h。     

Protein Adsorption on Erythrocytic Membranes and Its Effect on Erythrocyte Rheology in Athletes during Competition Exercise

The adsorption of high-molecular-weight plasma proteins on erythrocyte membranes was studied in athletes after prolonged exercise under competition conditions. The adsorption of individual high-molecular-weight protein fractions depended on their concentration. The adsorption index changed biphasically at submaximum exercise. The adsorption of plasma high-molecular-weight protein fractions was associated with the fluidity of concentrated erythrocyte suspensions. The adsorbed high-molecular-weight globulins and fibrinogen had different effects on the parameters of erythrocyte rheology.     

732树脂吸附蛋白质的机理研究

通过树脂吸附水溶液中蛋白质的试验 ,研究了 732树脂对蛋白质吸附过程机理 ,初步分析了动力学行为 ,包括吸附等温线方程、吸附速率方程、总传质系数、树脂内的有效扩散系数等     

PKC-Dependent Long-Term Effect of PMA on Protein Cell Surface Expression in Caco-2 Cells

Several recent data indicate that protein traffic is under the control of different phosphorylation pathways. In previous works, we have shown that cell surface expression of apical hydrolases and of a basolateral protein, “525” antigen, was impaired in Caco-2 cells treated with forskolin, a potent PKA activator (L. Baricaultet al.,1995,J. Cell Sci.,108, 2109–2121). Surprisingly, in these experiments forskolin did not seem to act through PKA activation. These cAMP-independent effects of FK may rely on cross-talk between intracellular phosphorylation pathways as described recently for PKA and PKC pathways. Therefore, we tested the hypothesis that PKC activation may induce effects comparable to those of FK on three brush border hydrolases as well as on 525 antigen cell surface expression in Caco-2 cells. Using enzymatic activity measurements and pulse–chase experiments combined with cell surface biotinylation assays, we show that long-term treatment with phorbol 12-myristate 13-acetate (PMA) impairs the overall expression of neither brush border hydrolases nor that of the 525 antigen but decreases total cell surface expression of these proteins. The apical and basolateral delivery pathways are equally affected. Using confocal laser scanning microscopy we show that the DPP IV and the 525 antigen that were not recovered from the cell surface were sequestrated in Lamp-1-positive lysosomal-related vesicles. PMA stimulates PKC translocation even after a 3-week treatment and induces PKC? redistribution to a vesicular- and membrane-associated compartment also labeled with cytokeratins. These results demonstrate that PMA-dependent PKC activation strongly impairs protein cell surface targeting. They also suggest that these PKC-dependent effects which are similar to those previously obtained with FK are relevant to the described cross-talk between PKA- and PKC-dependent phosphorylation pathways.     

Effect of Peptone on Azotobacter Morphology

Pleomorphism in cultures of Azotobacter vinelandii was induced by addition of Difco peptone to the growth medium. Under the conditions of the study, 5% peptone gave rise to transient forms described as "fungoid cells" which were osmotically fragile. After some 48 hr of culture, they became osmotically stable and resumed a more typical morphology. It was shown that the pleomorphism-inducing principle in peptone was glycine.     

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ^1-6, the unfolding of proteins ⁷, and binding affinity of different ligands ⁸. By coupling these nanopores to the resistive-pulse technique ^9-12, such measurements can be done under a wide variety of conditions and without the need for labeling ³. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions ¹³. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.     

Nanoneedle Surface Modification with 2-Methacryloyloxyethyl Phosphorylcholine Polymer to Reduce Nonspecific Protein Adsorption in a Living Cell

Previously, we developed a new molecular delivery system to target single living cells by using atomic force microscope and ultrathin needle referred to as nanoneedle. This system delivers molecules into the cell by attaching them to the surface of nanoneedle. However, nonspecific protein adsorption on the nanoneedle surface inside the living cells limits the range of application of this system. In the present study, we focused on nonspecific protein adsorption onto the nanoneedle surface inside the cells and examined whether this protein adsorption was reduced by modifying the nanoneedle surface with a biocompatible phospholipid polymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC) unit. MPC polymer coating of the surface of silicon wafer reduced nonspecific adsorption of proteins from liver extracts and prevented the formation of clot-like protein aggregates. MPC polymer also decreased nonspecific adsorption of cytosolic protein onto the nanoneedle surface inside the living cell. On the other hand, MPC polymer showed no effect on nonspecific mechanical interaction between nanoneedle and the cell components. Surface modification with MPC polymer is a useful technique to modify the surface properties of nanoneedle.     

Cell Surface Morphology After Trypsinisation Depends on Initial Cell Shape

Cells growing in tissue culture exhibit constant variation in shape and surface morphology, particularly during the process of mitosis, where the cell rounds up exhibiting an intensely microvillous surface prior to cytokinesis. During routine subculturing, cells are induced to round up and relinquish contact with the substratum. Although the cells retain their viability throughout trypsinisation, their surface morphology demonstrates a variety of changes between finger-like microvillous projections, and spherical protruberances termed blebs.
The reaction of individual cells to cell rounding, in the presence of trypsin appears to be dependent on cell shape, which may be modulated naturally or altered by experimental agents. Cells of bipolar morphology, termed fibroblasts, produce a blebbed surface morphology in response to trypsin, whereas isometric, 'epithelioid' cells respond by the formation of a microvillous cell surface.
Blebbed cells subsequently undergo membrane reorganisation towards a more organised, and more permanent microvillous cell surface, even in the continued presence of trypsin. Naturally occurring spherical cells, for example, mitotic or suspension cultures, are microvillous and trypsin has no effect on their surface morphology. It would appear that blebs are the cells response to experimentally induced rapid change of shape of well spread cells, and thus represent a pathological response for prevention of membrane loss in conditions which produce a rapid assumption of a minimum surface area configuration, i.e. a sphere, which occurs too quickly for membrane resorption, or normal storage in the form of microvilli.