【作者】 刘静；

【导师】 牟伯中； 邹爱华；

【作者基本信息】 华东理工大学 ， 物理化学， 2010， 博士

【摘要】 表面活性素由脂肪酸链和肽链组成,是脂肽中的一类同系物,具有良好的表面活性和生物活性。与化学合成表面活性剂相比,表面活性素具有环境友好、生物可适应性等优点。脂肪酸链碳数为15的表面活性素(surfactin-C15)是表面活性素同系物中的主要组分。本文研究了surfactin-C 15在溶液中的性质及其与生物大分子之间相互作用,主要包括以下内容。第一部分：Surfactin-C15在pH 7.4磷酸盐缓冲液(PBS)中的性质研究。采用表面张力、小角中子散射、冷冻蚀刻透射电镜、Langmuir-Blodgett (LB)膜以及圆二色谱等方法深入地研究了surfactin-C 15的聚集行为。Surfactin-C 15的CMC为1.54×10-5M,CMC处的表面张力为27.7 mN／N,在气／液界面处单分子面积为107.8 A2。Surfactin-C 15分子在溶液中为β-折叠构象,使得surfactin-C 15在较低的浓度时具有很好的表面活性。小角中子散射和冷冻蚀刻透射电镜结果说明surfactin-C 15在较低浓度时具有很强的自聚能力,形成球状胶束和一些较大的聚集体,其球形胶束的聚集数(<20)要远小于相似烷基链长度的传统的表面活性剂的聚集数。第二部分：Surfactin-C 15与蛋白质的相互作用。采用表面张力法、圆二色谱、小角中子散射、荧光法、冷冻蚀刻透射电镜等方法研究了表面活性素与几种典型蛋白质(牛血清蛋白、牛血红蛋白、溶菌酶)的相互作用。研究发现surfactin-C 15与不同种类的蛋白质之间的相互作用主要通过静电作用、疏水作用、分子之间的氢键来实现,并受蛋白质的结构、所带电荷及溶液pH的影响。随着surfactin-C 15浓度的增加,不仅能够使得蛋白质紧密的结构展开最终发生变性形成珍珠链结构；而且能够改变蛋白质的二级结构,使得蛋白质分子结构中的α螺旋结构含量减少。与传统的表面活性剂相比,在相似的浓度区间surfactin-C 15对蛋白质具有温和的破坏作用。第三部分：以卵磷脂形成的脂质体作为生物膜模型,通过利用动态光散射法、冷冻蚀刻透射电镜等方法对脂质体与surfactin-C 15之间的相互作用进行了细致研究,随着surfactin-C 15浓度的增加,采用动态光散射法得到脂质体的水力学直径从～120 nm下降到～10 nm,冷冻蚀刻透射电镜观察到从脂质体到卵磷脂／surfactin-C 15混合胶束的结构变化。这个结果也通过体系的透射率和微极性的下降来证实。从脂质体到卵磷脂／surfactin-C 15混合胶束的动态变化也被动态光散射法记录下来,表明脂质体在surfactin-C 15溶液中的泄漏和破坏过程能够在很短的时间内完成。第四部分：Surfactin-C 15与药物分子的相互作用。研究了甲苯胺蓝在surfactin-C 15溶液中的聚集特性和分配行为。通过紫外可见光谱和荧光探针,可以发现甲苯胺蓝分子定位在surfactin-C 15胶束中的栅栏层,这将有利于甲苯胺蓝聚集体的形成。Surfactin-C 15胶束中甲苯胺蓝的定位是由于甲苯胺蓝分子中二甲基氨基和表面活性素氨基酸残基的静电作用。甲苯胺蓝和surfactin-C 15胶束的结合常数和甲苯胺蓝在胶束相和水相中的分配系数通过Benesi-Hildebrand和相分离模型计算得到,结果显示,甲苯胺蓝在surfactin-C 15胶束中的定位时,静电吸引起了重要的作用。更多还原

【Abstract】 Surfactin is a kind of lipopeptide containing seven amino acid residues and aβ-hydroxy fatty acid residues, which has excellent surface properties and biological activities. Compared with the chemical surfactants, surfactin has some unique advantages such as environment friendly, higher biodegradability. Surfactin isoform (surfactin-C15) with aβ-hydroxy fatty acid chain of 15 carbon atoms is the primary ingredient in surfactin variants. In this paper the aggregation activity of surfactin-C15 and the interaction between surfactin-C15 and biomacromolecule were investigated. The results include as follows.In the first section, surface tension, small-angle neutron scattering (SANS), freeze-fracture transmission electron microscopy (FF-TEM) and circular dichroism(CD) measurements have been used to study the self-aggregation properties of surfactin-C 15 in 0.01 M (pH 7.4) phosphorate buffer solution (PBS). It has been found that critical micelle concentration (CMC) is 1.5×10-5 M, the surface tension is 27.7 mN/m and the area per molecule at air-water interface is 107.8 A2 for surfactin-C 15. surfactin-C15 molecules adopt aβ-sheet conformation making it Surface-active at such low concentrations. From SANS and FF-TEM results it is shown that surfactin-C 15 exhibits strong self-assembly ability to form spherical micelles and some larger aggregates even at the rare low concentration. The aggregation number of spherical micelles (<20) is much smaller than that of conventional surfactants with similar alkyl chain length.In the second section, we studied the interaction of surfactin-C 15 with several representative proteins in PBS solutions by UV-vis spectra, fluorescence spectra, SANS, AFM, FF-TEM and DLS measurements. It is found that the interactions between surfactin-C 15 and different protein species are mainly due to the electrostatic attraction, hydrophobic interaction and hydrogen bonds, which effected by the different structure and charge of protein and the solution environment (pH). With the increase of surfactin-C 15 concentration, the interactions between surfactin-C 15 and different protein lead to the unfolding of protein and take on a’necklace model’microstructure. Additionally, surfactin-C 15 can change the secondary combined with the decrease in a-helix content. Compared to the traditional surfactant, surfactin-C 15 has the gentle disruption effect on the structure of protein and presents different physicochemical behaviors.In the third section:surfactin-C 15 effect on Phosphatidylcholine (PC) liposome (model membrane) was studied by fluorescence, atomic force microscopy (AFM), FF-TEM and dynamic light scattering measurement (DLS). DLS results showed that the hydrodynamic diameter of PC liposome decreased with the surfactin-C 15 addition, which was verified by the decrease of transmittance and micropolarity of this system. These results were because of the microstructure change of PC liposome to PC/surfactin-C 15 mixed micelles. And it is interesting that the dynamic change of PC liposome to PC/surfactin-C15 mixed micelle was recorded by DLS and was further conformed by FF-TEM images. In sum, the process of PC liposome to PC/surfactin-C 15 micelle is the solubilization of PC liposome and the reorganization of PC/surfactin-C 15 aggregates.In the forth section:Aggregation properties and distribution behavior of toluidine blue (TB) in surfactin-C 15 solution have been investigated. It was detected by UV-vis spectra and fluorescence probe that TB can be located in the palisade of surfactin-C 15 micelle, which facilitates the formation of TB aggregates. This is because the dimethylamino group in TB molecule is prone to interact with the two negatively charged amino acid of surfactin-C 15 molecule. The binding constants of TB with surfactin-C 15 micelles and the distribution coefficients between the micelles phase and the aqueous phase have been calculated by Benesi-Hildebrand method and phase separation model, respectively. The values of thermodynamics functions show that electrostatic attractive interaction plays a dominating role at the location of TB in surfactin-C 15 micelles.更多还原