Crystallization and stability of different protein crystal modifications: A case study of lysozyme

The crystallization, including both the phase diagram and the phase transition of hen egg white lysozyme (HEWL) was investigated. A tetragonal modification and a needle modification were obtained during crystallization. The phase diagram and stability of two modifications in both acid and basic pH solutions (pH 4.5, 8.0 and 9.0) were determined. Besides in acid solutions, the well‐known tetragonal crystals can also be obtained in basic solutions at low temperature (7 °C) while the needle like modification can only crystallize in a basic solution. Based on the phase diagram, phase transfer behavior was found to exist between the two modifications. In basic solutions, tetragonal modification can transfer to needle shaped crystals. This process can be affected by a changing of pH and temperature. While in acid buffer, the needle shaped crystals dissolve and tetragonal crystals crystallize and remain in solution.     

2-巯基苯并咪唑与溶菌酶作用机理的研究

在生理条件下利用光谱法和分子模拟技术研究了2-巯基苯并咪唑对溶菌酶的毒性作用机理,分析了二者的结合特性,探讨了溶菌酶空间结构和酶活性的变化,模拟了二者的具体结合位置,结果表明2-巯基苯并咪唑可以通过静态猝灭的方式显著地猝灭溶菌酶的内源荧光。通过测量不同温度下的结合位点数、结合常数以及热力学常数,显示2-巯基苯并咪唑与溶菌酶主要通过氢键和范德华力相结合。分子模拟结果显示2-巯基苯并咪唑结合在溶菌酶的活性位点处,并最终导致溶菌酶空间结构和酶活性的变化。该研究为从分子水平上考察2-巯基苯并咪唑的毒性作用机理提供了参考。     

N-磷酰化肽酯及小肽与溶菌酶相互作用的ESI-MS研究

用ESI-MS研究了一系列结构具有可比性的N-磷酰化肽酯及小肽和溶菌酶的非共价相互作用, 比较了磷酰化肽酯及小肽分子中的不同基团对相互作用的影响. 结果表明—OH对其与溶菌酶的相互作用有较大贡献; 芳香环由于位阻原因, 对相互作用有促进和阻碍双重效应; 当—OH与芳香环相连时会发生协同效应, 可使相互作用显著增强. 磷酰化肽酯及小肽的体积大小、空间位阻对相互作用亦有显著影响. 磷酰化二肽中氨基酸残基的构型、顺序、碳链长短的变化(增加1～2个C)对其与蛋白溶菌酶之间的相互作用在质谱中没有表现出影响. 分子结构较为伸展、分子柔顺性好、空间位阻较小的磷酰化小肽更容易使蛋白在溶液中的构象趋于收缩, 而构象较为收缩的蛋白分子更易结合空间位阻较小的磷酰化小肽分子.     

The role of peptides and proteins in melanoidin formation

High‐molecular‐weight (HMW) coloured compounds called melanoidins are widely distributed, particularly in foods. It has been proposed that they originate through the Maillard reaction, a non‐enzymatic browning reaction, due to the interaction between protein or peptide amino groups and carbohydrates. The melanoidin structure is not definitively known, and they have been generally defined as HMW nitrogen‐containing brown polymers. In order to gain information on the nature of melanoidins, a simple in vitro model was chosen to investigate the products of the reactions between sugars and peptide/proteins. This approach would elucidate whether melanoidin formation is due to the binding of different sugar units to a peptide/protein or vice versa. With this aim, the reactivity of two different peptides, EPK177 and physalaemin, and a low‐molecular‐weight (LMW) protein, lysozyme, was tested towards different saccharides (glucose, maltotriose (MT), maltopentaose and dextran 1000) in aqueous solutions at different temperatures. The incubation mixtures were analysed at different reaction times by MALDI/MS. Furthermore, in order to verify the possible role of sugar pyrolysis products in melanoidin formation, the products arising from the thermal treatment at 200 °C of MT were incubated with lysozyme, and the reaction products were analysed by the same MS approach. The obtained results allowed the establishment of some general views: melanoidins cannot simply originate by reactions of sugar moieties with proteins. In fact, the reaction easily occurs, but it does not lead to any coloured product, as melanoidins have been described to be; melanoidins cannot originate from the thermal degradation products of glycated proteins. In fact, the thermal treatment of glycated lysozyme leads to a severe degradation of the protein with the formation of LMW species, far from the view of melanoidins as HMW compounds; experimental evidence has been gained on the melanoidin formation through reaction of intact protein with the pyrolysis products of MT. This hypothesis has been supported either from MALDI measurements or from spectroscopic data that show an absorption band in the range 300–600 nm, typical of melanoidins. Copyright © 2009 John Wiley & Sons, Ltd.     

用蛋白电泳和高效凝胶排阻色谱法分析还原脲变性蛋白溶菌酶稀释复性过程的集聚体

本文利用蛋白电泳和高效凝胶排阻层析法分析了还原脲变性蛋白溶菌酶稀释复性过程中的集聚体。当用复性液稀释复性还原脲变性蛋白溶菌酶时,会迅速产生可观量的沉淀。沉淀和上清液的不连续十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和高效凝胶排阻层析分析结果表明,还原脲变性蛋白溶菌酶在稀释复性过程中除了能够复性成天然态蛋白溶菌酶分子外,还会形成可溶的蛋白溶菌酶分子二聚体和三聚体,二聚体和三聚体主要是靠分子间二硫键的错配连接而成的;可溶的蛋白溶菌酶分子二聚体之间通过非共价键相互作用而形成集聚体沉淀,而可溶的三聚体溶菌酶分子则仍处于复性液上清液中。     

Thermochemical, volumetric and spectroscopic properties of lysozyme–poly(ethylene) glycol system

Investigations of lysozyme–polyethylene glycol system were made by differential scanning calorimetry, fluorescence and density techniques. The values of unfolding enthalpies, ΔH_N^U, unfolding temperatures, T_m, excess molar heat capacities, ΔC_p, and apparent molar volumes, V_Φ , were determined as functions of PEG concentration. The three PEGs of average molecular mass (MW) 6000, 10000, 20000 were used as macromolecular crowding agents. The concentration of polymers was changed in the range 0–30% mass per volume (w/v). The values of ΔH_N^U remained constant with no dependence on PEG concentration, while PEG addition to buffered lysozyme solutions caused linear decrease of T_m. The values of ΔC_p and V_Φ of lysozyme dramatically changed in the range of 8–10% of PEG concentration. The fluorescence spectroscopy was used in order to investigate the polymer influence on possible solvent–lysozyme interactions. The electrical properties of polymer–water and polymer–buffer systems, the dielectric constants of solutions were determined with use of impedance spectroscopy.     

Interaction of a homologous series of <Emphasis Type="Italic">n</Emphasis>-alkyl trimethyl ammonium bromides with eggwhite lysozyme

The interaction of a series of n-alkyl trimethyl ammonium bromides (C₁₂, C₁₄ and C₁₆) with egg white lysozyme have been studied using fluorescence and UV-Vis spectroscopies and isothermal titration calorimetry (ITC). The trend of variation of molar absorptivity at 281 nm, quantum yields (λ_ex=281 nm) and heat of interaction with respect to surfactant concentration, were measured. The spectrophotometric results show that the hydrophobic interactions have a major role in denaturation mechanism and it would be increased with increasing in hydrocarbon tail length of surfactant. The ITC results indicated the two-step mechanism for unfolding of lysozyme due to its interaction with surfactants.     

茜素红S-铜(Ⅱ)金属配合物与溶菌酶作用研究

采用UV-Vis光谱法研究了pH=4.25的缓冲溶液中茜素红S(ARS)-铜(Ⅱ)金属配合物与溶菌酶(LYS)的结合反应。提出了双波长物质的量比法和平衡透析物质的量比法，与单波长物质的量比法进行对照测定研究，实验结果基本相符。研究发现，ARS-Cu(Ⅱ)-LYS的最大吸收波长为526 nm，比ARS红移96 nm，比ARS-Cu(Ⅱ)配合物红移10 nm。在526 nm处，测得ARS-Cu(Ⅱ)-LYS三元配合物的结合比为n_ARS∶n_Cu(Ⅱ)∶n_LYS=6∶3∶1，摩尔吸光系数ε=7.93×10⁴ L·mol^-1·cm^-1，ARS-Cu(Ⅱ)配合物与LYS作用的条件平衡常数K=3.21×10¹²。ARS-Cu(Ⅱ)与LYS之间的作用力为配位键和电荷力。     

Chemical modification of polymeric microchip devices

Analytical polymeric microchips in both fluidic and array formats offer short analysis times, coupling of many sample processing and chemical reaction steps on one platform with minimal sample and reagent consumption, as well as low cost, minimal fabrication times and disposability. However, the invariable bulk properties of most commercial polymers have driven researchers to develop new modification strategies. This article critically reviews the scope and development of chemical modifications of such polymeric chips since 2003. Surface modifications were based on chemical derivatization or activation of surface layers with reagent solutions, reactive gases and irradiation. Bulk modification of polymer chips used newly incorporation of monomers with selective chemical functionalities throughout the bulk polymer material and integrated the chip modification and fabrication into a single step. Such modifications hold a great promise for establishing a true ‘lab-on-chip’ as can be seen from many novel applications for modulating electroosmosis, suppressing protein adsorption in microchip capillary electrophoretic separations, extraction of analytes and for zone-specific binding of enzymes and other biomolecules.