Studies on Aggregation Interaction between Reduced Denatured Egg White Lysozymes during Refolding Procedure in Urea Solution

The aggregation interaction between reduced-denatured egg white lysozymes during refolding procedure in urea solution was studied by means of reducing and non-reducing protein electrophoreses. Results of non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE） of the supernatant and aggregate precipitate formed in refolding process show that except being refolded to native egg white lysozymes, the reduced-denatured lysozymes can also form the aggregates with molecular weights （MW） being separately about 30.0 and 35.0 kD, while the reducing SDS-PAGE and the refolding results in the presence of sodium dodecyl sulphate show that these aggregates are formed chiefly through the misconnection of disulfide bonds between the reduced-denatured lysozymes, and the aggregate precipitates are formed through the non-covalent interactions between the aggregates with molecular weight being about 30.0 kD. From the results of electrophoresis and size-exclusion chromatographic analyses, it can be inferred that the aggregates with molecular weights being about 30.0 and 35.0 kD are bi-molecular and tri-molecular egg white lysozyme aggregates, respectively. And finally, a suggested refolding mechanism of reduced-denatured egg white lysozymes in urea solution was presented.     

Partial Molar Volumes of Amino Acids and Peptides in Aqueous Salt Solutions at 25°C and a Correlation with Stability of Proteins in the Presence of Salts

Partial molar volumes for a homologous series of amino acids and peptides have been measured in aqueous 1M sodium acetate, sodium thiocyanate, and sodium sulfate at 25^°C. These data have been utilized in conjunction with the data in water to deduce partial molar volumes of transfer V _2,m ⁰(tr) from water to these aqueous salt solutions. The volumes of transfer for the amino acids and peptides are found to be positive. The interpretation is that this result arises from the dominant interaction of the sodium salts with the charged centers of amino acids and peptides. Thermal denaturation of the structurally homologous proteins lysozyme and -lactalbumin has been studied in the presence of these salts. Significant thermal stabilization of hen egg-white lysozyme has been observed in the presence of sodium acetate and sodium sulfate. However, the thermal stabilization observed for -lactalbumin is very small in the presence of these salts and sodium thiocyanate leads to a lowering of its thermal denaturation temperature. The rise in the surface tension of aqueous salt solutions with salt concentration has been correlated with the calorimetric and volumetric measurements. The results show that V _2,m ⁰(tr) depends less on the type of electrolyte than on the ionic strength of the solution. The V _2,m ⁰(tr) values correlate very well with the increase in the surface tension of aqueous salt solutions, indicating significant role of surface tension in interactions of amino acids, peptides, or protein with the salts.     

多孔导电膜刻蚀方法的改进及其在毛细管电泳蛋白进样浓缩中的应用

通过控制电压偏置方向,利用电渗流效应使毛细管多孔导电膜的刻蚀成功率得到显著提高。在刻蚀过程中于管中灌注偏碱性溶液,并在毛细管进口端与刻蚀池间施加正电压偏置。当刻蚀达到电通透时,在电渗流的作用下刻蚀自动停止,从而避免了过刻蚀。在含有2%～4%的葡聚糖的0.05mol/LTris-H3PO4(pH2.5)缓冲体系中,利用该多孔膜对溶菌酶和牛血清白蛋白实现了接近理论值的浓缩倍率。对酸碱性不同的蛋白浓缩效率的差别进行了讨论。利用电进样浓缩30min使常规光度检测器(214nm)对两个蛋白样品的检出限减小为1×10-11mol/L。     

超声喷雾电离源的研制与表征

研制了超声喷雾电离源(SSI)。采用核糖核酸A、溶菌酶等样品和商品化的线性离子阱质谱仪对该电离源进行了表征。实验发现,对于生物大分子,超声喷雾电离质谱(SSI-MS)能够获得与电喷雾质谱(ESI-MS)类似的多电荷离子。但与同等条件下ESI-MS所获得的谱图相比,SSI主要获得低价态的多电荷离子。在此基础上,系统考察了SSI-MS各主要操作参数对不同价态蛋白质多电荷离子信号强度的影响,并提出了SSI离子化的可能机理。结果表明,在喷雾气压3.4~3.6 MPa、喷雾口到质谱入口的距离4~6 mm、离子传输管温度250~300℃、样液流速50~100μL/min、2%~5%甲酸酸性且不含甲醇的条件下,各价态蛋白质离子信号强度及信号分布均达到最优;而离子传输管温度越高,喷雾压力越大或溶剂中甲醇等挥发性成分越高,则越有利于低价态离子的形成。     

Direct Observation of Non-covalent Complexes for Phosphorylated Flavonoid-protein Interaction by ESI

Diethyl flavon-7-yl phosphate was synthesized by modified Atheron-Todd reaction. The result of ESI shows that the phosphated flavonoids possess stronger binding affinities toward proteins such as myoglobin, insulin and lysozyme and are easier to form the non-covalent complexes with them.     

高效阳离子交换法分离纯化蛋清中的溶菌酶

 建立了用高效阳离子交换分离纯化蛋清中溶菌酶的新方法 ,讨论了纯化的工艺条件。蛋清样品匀浆后 ,用氯化钠初步纯化 ,然后用弱阳离子交换柱XIDACE WCX分离。结果表明 ,被纯化的溶菌酶和杂蛋白得到很好分离。经活性检测 ,溶菌酶过柱后的活性回收率为 10 7% ,蛋白的比活为 15 4 6 7U/mg ,纯化倍数提高了 5 6倍。用尺寸排阻 (SEC)鉴定 ,得到的溶菌酶呈均一性。该法较传统软基质低压离子交换分离速度快 ,纯化效率高。     

Lysozyme coated DNA and DNA/SWNT fibers by solution spinning

DNA fibers were prepared by solution spinning of DNA in a lysozyme (LSZ) coagulation/gelation bath. Strong positive charges carried by LSZ protein condensed the DNA (strong negative charged) molecules resulting in self‐assembly and the formation of fibrillar structures in a gel‐like network. DNA/LSZ fibril formation was found to be dependent on the ratio of DNA to LSZ. A minimum 0.1 wt.‐% of LSZ was necessary to condense 0.1 wt.‐% of DNA into micro‐fibrils. Macroscopic fiber spinning was possible by introducing a 0.1 wt.‐% DNA aqueous solution into a 0.2 wt.‐% LSZ coagulation bath which resulted in fibers with ≈20 µm diameter. Single‐walled carbon nanotubes (SWNT) were also incorporated into these fibers to explore the possibility for creating hybrid materials. All DNA‐based fibers exhibit strong birefringence confirming molecular orientation along the fiber axis. Due to the presence of LSZ, the fibers exhibit antimicrobial activity against bacteria like Micrococcus lysodeikticus.

     

Macromolecular crystallography research at Trombay

Neutron diffraction studies of hydrogen positions in small molecules of biological interest at Trombay have provided valuable information that has been used in protein and enzyme structure model-building and in developing hydrogen bond potential functions. The new R-5 reactor is expected to provide higher neutron fluxes and also make possible smallangle neutron scattering studies of large biomolecules and bio-aggregates. In the last few years infrastructure facilities have also been established for macromolecular x-ray crystallography research. Meanwhile, the refinement of carbonic hydrases’ and lysozyme structures have been carried out and interesting results obtained on protein dynamics and structure-function relationships. Some interesting presynaptic toxin phospholipases have also been taken up for study.     

Photoinduced electron transfer between hen egg white lysozyme and anticancer drug menadione

The protein, hen egg white lysozyme, on photoexcitation undergoes electron transfer with menadione (2-methyl-1,4-naphthoquinone), a widely known anticancer drug. With the addition of menadione the fluorescence of lysozyme is quenched with the simultaneous formation of an excited state charge-transfer complex in the longer wavelength and a ground state complex. The former is further evident from laser flash photolysis studies, which indicate a tryptophan to menadione electron transfer. From fluorescence quenching studies the binding constant is found to be ∼1.7×10⁴ M⁻¹ with the corresponding changes in enthalpy (ΔH°) and entropy (ΔS°) as 12.24 kJ mol⁻¹ and 124.12 J mol⁻¹ K⁻¹, respectively, indicative of an entropy-driven process. The circular dichroism studies also show some structural changes with increase in α-helical content in the protein on interaction with menadione. Finally, docking studies give some insight into the role of Trp 108 of lysozyme in the interaction.     

Antifungal activity,DNA and lysozyme binding affinity of Pd(II) and Pt(II) complexes bearing N,N-pyridylbenzimidazole ligand

Abstract

The lysozyme- and DNA-binding affinities of the biologically active cisplatin analogues, Pd(II) (1) and Pt(II) (2) complexes of functionalized N,N-pyridylbenzimidazole ligand, are reported. The electronic transitions were studied by time-dependent density functional theory. Complex 1 exhibits interesting antifungal activity against Candida albicans (MIC?=?32?μg?mL^?1; 64?nM) and Cryptococcus neoformans (MIC?=?16?μg?mL^?1, 32?nM). Complex 1 is covalently bound to DNA and lysozyme via the elimination of the chloride ligand(s). When complex 2 interacts with lysozyme, two adduct peaks are observed in the mass spectrum corresponding to binding of Pt(II) ion to lysozyme.