电导法研究表面活性剂与蛋白质的相互作用

较系统地研究了溶液中离子型表面活性剂与蛋白质相互作用时电导率的变化,并根据实验现象,得出表面活性剂与蛋白质作用的两种模式－疏水作用模式和电荷作用模式。表面活性剂采用疏水作用模式与蛋白质结合时,蛋白质的二硫键逐一断裂,三级结构逐渐打开,电导率曲线出现一些小“平台”,采用电荷作用模式吸附蛋白质时,首先形成疏水复合体,产生白色浑浊,随离子型表面活性剂浓度的增加,疏水复合体转变成亲水复合体,白色浑浊     

AOT/异辛烷反胶束萃取纯化胰蛋白酶及酶变性失活问题的解决

用反胶束技术分离纯化蛋白质,具有高选择性、易于大规模操作等优点,具有良好的工业应用前景。但是离子型表面活性剂形成的反胶束体系萃取蛋白质容易引起蛋白质的变性,这是由于离子型表面活性剂的强电荷作用所导致的。对用AOT/异辛烷反胶束体系从胰酶粗提物中萃取胰蛋白酶进行了研究,通过在反胶束相加入乙醇,解决了反胶束萃取蛋白质时蛋白质变性失活的问题。并且由于乙醇的加入大大减少了分相的时间,简化了实验步骤,优化了实验方法,使此技术在工业上的大规模应用成为可能。通过优化各种实验条件,胰蛋白酶的前萃取率达到90%,反萃取率接近100%。最终得率为88%。纯化后的比活提高了5倍多,从300U/mg左右提高到了1800U/mg。     

用乙醇消除AOT/异辛烷反胶束体系萃取过程中蛋白质的变性失活

用反胶束技术分离纯化蛋白质,具有高选择性、易于大规模操作等优点,具有良好的工业应用前景。但是离子型表面活性剂形成的反胶束体系萃取蛋白质容易引起蛋白质的变性,这是由于离子型表面活性剂的强电荷作用会导致蛋白质发生变性,从而在两相界面上产生沉淀。这也是离子型反胶束体系用于蛋白质萃取所存在的最大的困难。本文对用AOT/异辛烷反胶束体系从胰酶粗提物中萃取胰蛋白酶进行了研究,通过在反胶束相加入乙醇,解决了反胶束萃取蛋白质时使蛋白质变性失活的问题,并且大大减少了分相的时间。前萃取和反萃取之后的分相时间只需要10分钟左右,简化了实验步骤,优化了实验方法,在工业上的大规模应用成为可能。在本研究中,胰蛋白酶的前萃取率达到90%,反萃取率接近100%。最终得率为88%。纯化后的比活提高了5倍多,从300U/mg左右提高到了1800U/mg。     

ctDNA与离子型表面活性剂相互作用的共振瑞利散射法研究

利用共振瑞利散射光谱(RRS)方法研究了ctDNA与离子型表面活性剂的相互作用.结果表明:阳离子表面活性剂通过静电、疏水等作用在ctDNA表面聚集,造成ctDNA的RRS大大增强;阴离子表面活性剂对吖啶橙(AO)与ctDNA的相互作用有协同作用,能使AO-ctDNA的RRS信号大大增强.     

生物表面活性剂在提高原油采收率方面的应用

生物表面活性剂和一般的化学表面活性剂一样,都拥有亲水和疏水基因,是微生物生长在水不溶的有机物中并以营养物而产生的代谢产物。在油田应用中,生物表面活性剂的作用是微生物提高采收率的重要机理之一,具有水溶性好、反应产物均一、安全无毒、驱油效果好等特点。本文从产生生物表面活性剂的菌种及生物表面活性剂的类型、生物表面活性剂的特性、实验研究、矿场实验及展望等五个方面综述了生物表面活性剂在提高原油采收率方面的应     

几种二价阳离子及表面活性剂对元麦叶原生质体电泳率的影响(简报)

原生质体电泳率的研究已应用于了解质膜表面的电荷特性。Ca~( )能影响原生质体质膜表面的电荷状况,在诱导融合时起重要作用。关于二价阳离子对质膜功能的影响,Caldwell报道了Ca~( )、Mg~( )、Mn~( )等多种二价阳离子对质膜ATP酶不同的激活效应。此外,Helenius等指出表面活性剂能改变膜的结构,并与膜脂作用而引起溶胞。为进一步了解不同的二价阳离子对质膜电荷特性的影响,及表面活性剂在改变膜结构这一复杂过程中对质膜电性影响的状况,我们报道Ca~( )、Mg~( )、Mn~( )等二价阳离子及几种表面活性剂对原生质体电泳率影响的结果。     

表面活性剂与2,4-二硝基酚对小麦离体根的K~ 运转和盐呼吸的效应

表面活性剂对小麦离体根K~ 吸收和呼吸的抑制效应不同于解链剂DNP,表现在表面活性剂对小麦离体根K~ 吸收的抑制作用大于它对呼吸的抑制作用,和对KCI刺激的呼吸几乎没有影响;而DNP对小麦离体根呼吸的影响大于它对K~ 吸收的抑制作用,并几乎消除了KCI刺激的呼吸。看来两者通过不同的途径影响小麦离体根K~ 的吸收,DNP的抑制作用主要是氧化磷酸化解链的次生效应,而表面活性剂则可能直接对细胞膜和细胞膜的K~ 传递系统起作用。三种不同类型的表面活性剂对小麦离体根K~ 吸收显现出某些不同的效应,阳离子型表面活性剂氯化十六烷基三甲基铵(CTMA)和非离子型表面活性剂聚氧乙烯山梨醇月桂酸酯(Tween-20)在低浓度时对小麦离体根K~ 吸收有促进作用,阴离子型表面活性剂十二烷基硫酸钠(SDS)没有这种表现。这种现象可能与表面活性剂带电荷的性质,和它们与膜的作用方式有关。对CTMA和SDS引起小麦离体根K~ 外流的溶液紫外吸收光谱检测的结果表明,有在260和280mμ呈现吸收峰的物质从根上掉下来,并与Folin酚试剂起呈色反应,这些物质可能与小麦离体根K~ 吸收能力的丧失有联系。     

环境保护上的应用

<正> 对从稀悬液中采收小球藻的一种胶体气态阿夫隆(CGA)弥散新技术做了研究。CGA是直径25微米的小气泡。每个泡被包在一个表面活性剂的液壳中。利用CGA的漂浮性形成藻一泡复合体井漂浮至液面。采用阳离子表面活性剂(氯小二烷基毗咤)形成CGA时,     

淋巴细胞膜脂质对膜蛋白结构与功能的影响

淋巴细胞膜质组成及膜生物物理特性的变化显影响质膜上某些蛋白的结构与功能,膜脂质与膜蛋白的相互作用涉及两所带电荷性质,蛋白质疏水氨基酸等,同时也与两种分子空间位阻,水化作用有关。     

蛋白质层析用离子交换和疏水作用层析介质的发展概况

层析是蛋白质纯化的关键技术之一 ,作为层析技术的核心———层析介质一直以来是层析技术研究的一个热点。近年来 ,越来越多的新型层析介质被开发出来 ,如粒度均匀的交联多糖、人工合成的大孔聚合物、触角型吸附剂、软胶包裹在硬胶表面等介质。主要介绍应用较为广泛的IEC和HIC介质的组成、特性及其在蛋白质纯化中的应用 ,还研究了与HIC技术相关的两种新技术 :亲硫层析和疏水电荷诱导层析 (HCIC) ,重点介绍了HCIC的介质及其应用 ,同时也讨论了在蛋白质纯化中应用的三相纯化策略 (富集、中间纯化和精制 )。结合我国的实际情况 ,就当前蛋白质纯化的离子交换和疏水层析介质面临的挑战和未来的发展进行讨论并提出了建议     

Interaction of alpha-amylase with n-alkylammonium bromides

The interaction of alpha-amylase with n-alkylammonium bromides above and below their critical micellar concentrations (cmc) has been studied in buffer at pH 7 and 10 by UV spectrophotometry, photon correlation spectroscopy and Doppler microelectrophoresis. This interaction produces a complex that is dependent on pH of the medium. This complex appears at surfactant concentrations below the cmc, which means that individual surfactant molecules can bind tightly to native alpha-amylase. The complex maintains its aggregation state when the concentration of surfactants with a hydrocarbon chain of 16 carbons increases, but not for surfactants of 12 and 14 carbons. Measurements of zeta-potential indicate the influence of electrostatic and hydrophobic forces. When the size of the aggregate is maximal, proteins are at their point of zero charge. In such conditions, Van der Waals forces and contacts between the alkyl chain and the hydrophobic core of the protein favour the formation of a larger aggregate.     

Investigation on the mechanism of crystallization of soluble protein in the presence of nonionic surfactant

The mechanism of crystallization of soluble, globular protein (lysozyme) in the presence of nonionic surfactant C8E4 (tetraoxyethylene glycol monooctyl ether) was examined using both static and dynamic light scattering. The interprotein interaction was found to be attractive in solution conditions that yielded crystals and repulsive in the noncrystallizing solution conditions. The validity of the second virial coefficient as a criterion for predicting protein crystallization could be established even in the presence of nonionic surfactants. Our experiments indicate that the origin of the change in interactions can be attributed to the adsorption of nonionic surfactant monomers on soluble proteins, which is generally assumed to be the case with only membrane proteins. This adsorption screens the hydrophobic attractive force and enhances the hydration and electrostatic repulsive forces between protein molecules. Thus at low surfactant concentration, the effective protein-protein interaction remains repulsive. Large surfactant concentrations promote protein crystallization, possibly due to the attractive depletion force caused by the intervening free surfactant micelles.     

Effect of the head-group geometry of amino acid-based cationic surfactants on interaction with plasmid DNA

The interaction between DNA and different types of amino acid-based cationic surfactants was investigated. Particular attention was directed to determine the extent of influence of surfactant head-group geometry toward tuning the interaction behavior of these surfactants with DNA. An overview is obtained by gel retardation assay, isothermal titration calorimetry, fluorescence spectroscopy, and circular dichroism at different mole ratios of surfactant/DNA; also, cell viability was assessed. The studies show that the surfactants with more complex/bulkier hydrophobic head group interact more strongly with DNA but exclude ethidium bromide less efficiently; thus, the accessibility of DNA to small molecules is preserved to a certain extent. The presence of more hydrophobic groups surrounding the positive amino charge also gave rise to a significantly lower cytotoxicity. The surfactant self-assembly pattern is quite different without and with DNA, illustrating the roles of electrostatic and steric effects in determining the effective shape of a surfactant molecule.     

Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model

The interactions of dodecyltrimethylammonium bromides (DTABs) with hen egg lysozyme have been investigated at pH = 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. DTAB interacts endothermically and activate lysozyme. The endothermicity of the lysozyme-DTAB interaction is in marked contrast to the exothermic interactions between sodium dodecyl sulphate (SDS) and lysozyme which have been attributed to specific binding between the anionic sulphate head groups and cationic amino acid residues. The enthalpies of interaction between the cationic surfactant (DTAB) and lysozyme are dominated by the endothermic unfolding of the native structure followed by an exothermic solvation of the lysozyme-DTAB complex by the addition of extra DTAB. A new direct calorimetric method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The extended solvation model was used to reproduce the enthalpies of lysozyme-DTAB interaction over the whole range of DTAB concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of DTAB, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The DTAB-induced denaturation enthalpy of lysozyme is 86.46 +/- 0.02 kJ mol(-1).     

Interaction of reduced lysozyme with surfactants: Disulfide effects on reformed structure in micelles

The reformation of secondary structure for unfolded, disulfide reduced hen egg white lysozyme (HEWL) upon interaction with surfactants was studied using CD, fluorescence and IR (infrared) techniques. Equilibrium CD studies showed that reduced HEWL when mixed with negatively charged surfactants, such as SDS (sodium dodecyl sulfate), gradually regains average helical structure to a level equivalent to that obtained for the oxidized form also in SDS, but both forms lose tertiary structure in such environments. This non-native structure recovery process begins with monomer surfactant interaction but at higher concentrations is in part dependent on micelle formation, with the helical fraction reaching its maximum value with each surfactant only above the CMC. Fluorescence changes were more complex, evidencing an intermediate state at lower surfactant concentration. With positively charged surfactants the degree of helicity recovered was less, and the intermediate state in fluorescence was not seen. Stopped flow dynamics studies showed the CD kinetics fit to two exponentials as did the fluorescence. The faster steps in CD and fluorescence detected kinetics appear to be correlated which suggests formation of an intermediate on rapid interaction of the micelle and protein. The second step then reflected attainment of a stable surfactant solvated state which attains maximum helicity and moves the Trps to a more hydrophobic environment, which may occur in independent steps, as the slower kinetics are not well correlated.     

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0–20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.     

DNA-surfactant interactions: coupled cooperativity in ligand binding leads to duplex stabilization.

The cooperative nature of interaction of cationic surfactants with short oligonucleotides leading to eventual stabilization of DNA duplexes is demonstrated. At submicellar concentrations and DNA:surfactant charge ratios of 0.2 to 0.8, the association of single chain (CTAB) and double chain (DOTAP) surfactants to oligonucleotides is initiated by electrostatic interaction of cationic ligands with polyanionic DNA that aligns the surfactant molecules on the DNA template. This is followed by binding of new surfactant ligands to the initial complex, driven cooperatively by the hydrophobic forces, leading to in situ formation of surfactant-bound and bare duplexes as separate species. These exhibit independent melting behaviour characterised by double transition in thermal UV profiles, with a higher T(m) for surfactant-DNA complexes. Understanding the cooperative binding of the cationic surfactants to the DNA described here may have implications for rational design of DNA binding drugs and DNA delivery systems.     

底物的分散和溶解对甾体微生物酶反应的影响

甾体的溶解是微生物转化反应的控制步骤。表面活性剂对憎水性物质的增溶作用是具有憎水内核胶团形成的直接结果。随表面活性剂溶液浓度的增加,胶团数目增加,甾体水溶性增强。复配型分散剂PSE－MGE应用于甾体转化体系,相对于单组分分散剂PSE而言,实现同一程度甾体水溶性增强所需的表面活性剂用量减少,对菌体生长的负面影响较小,有利于转化。超声与分散剂配合使用的效果优于分解剂单独使用的效果,且使用较强的超声声强（12.4W/cm^2）和较短的超声时间（5min）时效果最佳。     

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.