原位椭圆偏振术研究牛血清清蛋白在固/液界面的吸附

用原位椭圆偏振术系统研究了硅片表面因素及缓冲液环境因素对牛血清清蛋白在固/液界面吸附的影响。在生理条件下,疏水表面与亲水表面相比BSA吸附量较大。随着硅片表面电荷密度增加,BSA吸附量增加。BSA吸附量当体溶液pH值等于BSA等电点时达到最大。而随着体溶液离子强度增加,BSA吸附量亦上升。实验结果提示:除了熵驱动作用之外,硅片表面与BSA分子及BSA分子之间的静电作用在BSA吸附中起着十分重要的作用。     

Effects of topology, length, and charge on the activity of a kininogen-derived peptide on lipid membranes and bacteria

Effects of topology, length, and charge on peptide interactions with lipid bilayers was investigated for variants of the human kininogen-derived peptide HKH20 (HKHGHGHGKHKNKGKKNGKH) by ellipsometry, CD, fluorescence spectroscopy, and z-potential measurements. The peptides display primarily random coil conformation in buffer and at lipid bilayers, and their lipid interaction is dominated by electrostatics, the latter evidenced by higher peptide adsorption and resulting membrane rupture for an anionic than for a zwitterionic membrane, as well as by strongly reduced adsorption and membrane rupture at high ionic strength. At sufficiently high peptide charge density, however, electrostatic interactions contribute to reducing the peptide adsorption and membrane defect formation. Truncating HKH20 into overlapping 10 amino acid peptides resulted in essentially eliminated membrane rupture and in a reduced amount peptide charges pinned at the lipid bilayer. Finally, cyclic HKH20 was found to be less efficient than the linear peptide in causing liposome rupture, partly due to a lower adsorption. Analogous results were found regarding bactericidal effects.     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence

The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing were observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.     

One-step immunoassay for measuring protein concentrations in plasma, based on precipitate-enhanced ellipsometry

Standard enzyme immunoassays (EIAs) require washing steps to remove excess enzyme-antibody complexes. Such washing is laborious, lengthens assay time, and increases assay scatter. Recently, so-called precipitate-enhanced immunoassays (PEIAs) were introduced. Instead of color formation due to enzymatic conversion of a chromogenic substrate, this technique measures the rate of precipitate formation due to conversion of a substrate with a precipitating product. Such precipitation can be measured in the presence of active enzyme-antibody complexes in the buffer and no washing is required. In the present study this technique was used in a one-step PEIA, without washing steps, for the measurement of plasma concentrations of fatty-acid-binding protein. Horseradish peroxidase was used as tagging enzyme and diaminobenzidine as precipitating substrate. Precipitate formation was measured by ellipsometry. Assay time of the one-step PEIA was much shorter than that for an existing standard EIA. Test results can be obtained within minutes, depending on the sensitivity required. Assay precision of the one-step PEIA was better than that of the standard EIA. In the one-step assay, loss of surface-bound conjugate due to washing is prevented, which could explain part of the improved sensitivity compared to that of the two-step PEIA. More importantly, the presence of substrate-converting enzyme-antibody complexes in the buffer caused a large enhancement of precipitation.     

On the Adsorption of Human Acidic Proline-rich Proteins (PRP-1 and PRP-3) and Statherin at Solid/liquid Interfaces

The objective of the present study was to investigate the adsorption of PRP-1, PRP-3 and statherin to solid surfaces in terms of dependence on concentration, the presence of electrolyte and surface wettability. Time resolved in situ ellipsometry was used to determine the adsorbed amounts and adsorption rates of pure PRP-1, PRP-3 and statherin onto pure (hydrophilic) and methylated (hydrophobized) silica surfaces. The initial film build-up was fast and plateaus were reached within 10 min at all concentrations for both types of surfaces and all proteins. The observed adsorption and calculated diffusion rates of PRP-1, PRP-3 and statherin, respectively, indicated that the initial adsorption was mass transport controlled at low concentrations. At hydrophobic surfaces, isotherm shapes and adsorbed amounts were similar for PRP-1 and PRP-3, while statherin adsorbed to a higher extent. At hydrophilic surfaces only PRP-1 adsorbed substantially, while for PRP-3 and statherin adsorbed amounts were low. The presence of Ca 2+ ions in the phosphate buffer solution increased the adsorption of statherin and PRP-3 on hydrophobic surfaces, while PRP-1 was unaffected. On hydrophilic surfaces, all three proteins adsorbed in higher amounts in NaCl, compared to CaCl 2 at similar ionic strength. It is concluded that acidic PRPs (PRP-1 and PRP-3) and statherin readily form films on a variety of materials and solution conditions, showing that their functions may be fulfilled under a wide range of conditions.     

Adsorption of low-density lipoprotein,its oxidation,and subsequent binding of specific recombinant antibodies: An in situ ellipsometric study

Background

Low-density lipoprotein (LDL) particles accumulate in the arterial wall and become oxidized during atherogenesis, leading to the formation of atherosclerotic plaques. The major protein of the LDL particle, apolipoprotein B-100 (apoB-100), becomes fragmented during oxidation and a target for the immune system.

Methods

In this study we used in situ ellipsometry to monitor the adsorption of LDL to solid silica surfaces and the effects of oxidation on the structure of the adsorbed LDL layer. We additionally investigated the binding kinetics of two recombinant human antibodies with different specificities recognizing epitopes of apoB-100 in surface-bound native and CuCl₂-oxidized LDL (oxLDL). The latter process was studied by adsorbing LDL and then adding the antibody and CuCl₂ while continuously monitoring adsorbed amount and the thickness of the film. The molar ratios between the antibodies and surface-bound LDL and oxLDL were calculated from these data.

Results

Our results indicate that oxidation of surface-bound LDL induces swelling of the layer, accompanied by a slight desorption. We further found that both antibodies were able to recognize LDL and oxLDL in its adsorbed orientation. Quantitative information was obtained on the number of available binding sites on surface-bound LDL and oxLDL for these two antibodies.

General significance

Using ellipsometry for real-time monitoring of adsorption, in situ oxidation of LDL and binding of specific recombinant antibodies to surface-bound LDL, will open up possibilities to map different conformations and orientations of LDL in the adsorbed state.     

亲和素蛋白与含有生物素的支撑平面膜之间相互作用的研究

生物功能分子的二维有序化组装是分子工程学的重要内容。本文通过构建有生物素受体的人工模型膜,对亲和素与生物素这一具有极强亲和力的系统之间的特异性相互作用的某些制约因素进行了探讨．应用ＬＢ膜技术结合激先椭圆偏振术和表面等离激元谱技术,较深入地研究了蛋白质与脂单层膜发生的非特异性吸附,特异性结合以及结合的侧向空间位阻效应．实验结果表明,膜表面电荷对非特异性吸附的速率有很大的影响,而对最终的蛋白吸附量影响不大;非特异性的吸附可以通过二阶阳离子的脱附而去除;受体蛋白质与配体间的特异性结合受到侧向空间位阻效应的制约．     

亲和素蛋白与含有生物素的支撑平面膜之间相互作用的研究

生物功能分子的二维有序化组装是分子工程学的重要内容,本文通过构建有生物素受体的人工模型膜,对亲和素与生物这一具有极强亲和力的系统之间的特异性相互作用的某些制约因素进行了探讨,应用ＬＢ膜技术结合激光椭圆偏振术和表面等离激元谱技术,较深入地研究了蛋白质与脂单层膜发发的非特异性吸附,特异性结合以及结合的侧向空间位阻效应,实验结果表明,膜表面电荷对非特异性吸附的速率有很大的影响,而对最终的蛋白质附量影响不     

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.