表面活性剂与过氧化氢酶相互作用对酶活性和构象的影响
发布时间：2018-09-06 09:04
【摘要】：与很多合成的催化剂相比,酶催化具有较高的选择性和催化效率、较小的生物毒性及反应条件温和等优点,是生物体内各种生物化学过程必不可少的。在酶的应用中,总是要考虑两个基本问题,即酶在反应环境下的催化活性和活性的动力学稳定性。表面活性剂是酶催化反应体系中经常使用的添加剂,够调控酶的催化活性和稳定性。本论文采用等温滴定量热(ITC)、荧光光谱、圆二色谱和紫外可见光谱等方法,研究了阴离子、阳离子和两性离子表面活性剂与过氧化氢酶(BLC)的相互作用及对酶结构和活性的影响,从热力学与分子结构的角度讨论了表面活性剂对酶活性影响的机理。主要研究内容和结果如下:1.通过紫外可见光谱(UV-vis)表征了表面活性剂对酶活性的影响。结果表明,阴离子型表面活性剂(SDS、SDSo)在低浓度下能抑制BLC活性,并且在它们各自的临界胶束浓度(cmc)附近时能够使酶变性。当阳离子型表面活性剂(DTAB)逐渐加入到BLC溶液时,在DTAB浓度达到cmc以前,过氧化氢酶经历了聚集而后再分散的过程,酶的相对活性降低到80%左右,而动力学稳定性明显提高。两性离子型甜菜碱表面活性剂(SB3-12)对酶的活性没有明显的影响,对酶的天然结构也具有一定的保护作用。在SB3-12存在时,过量的SB3-12能抑制SDS对BLC活性的影响。发现当xSB3-120.5时,BLC依然能够保持较高的活性,当xSB3-120.5时,BLC的活性迅速降低,BLC的活性受两种表面活性剂的摩尔分数控制。当BLC在DTAB与SB3-12混合表面活性剂溶液中培养时,酶活性受DTAB浓度控制。2.采用等温滴定量热研究了表面活性剂与酶分子之间的相互作用。研究结果表明,表面活性剂与酶的相互作用的热力学与极性头基的电荷性质有关,离子型表面活性剂首先会与酶分子上的相反电荷位点结合。SDS与酶之间的静电作用伴随着烷基链与酶分子的疏水作用,过量的SDS在酶分子表面形成分子簇,使酶展开;当DTAB与酶发生静电作用时,由于酶表面的负电荷位点附近缺少疏水域,过量的DTAB在不同酶分子上形成的分子簇能够聚集成共享胶束,促进了BLC分子的靠近并发生聚沉。随着DTAB浓度的增加,共享胶束解离成结合在酶表面上的半胶束,增加的静电排斥效应使聚集体开始重新分散;两性离子的SB3-12与酶分子之间的相互作用要比DTAB或SDS与酶分子之间的相互作用都要弱。SB3-12和SDS形成的混合表面活性剂与酶的相互作用由它们的摩尔分数控制,当xSDS0.50时,SDS分子协同地与酶分子作用形成分子簇,同时也与SB3-12胶束作用形成混合胶束,在一定的组成区间能够抑制SDS对BLC的变性效应。当xSDS0.50时,过量的SDS作用于BLC分子,使酶分子展开。对于SB3-12/DTAB/BLC混合体系,SB3-12不影响DTAB与BLC的静电作用,但是能够促进BLC分子之间形成混合的或SB3-12和DTAB分别构成的共享胶束,减小了使BLC聚沉的DTAB浓度。3.采用荧光光谱法和圆二色谱法研究了不同类型的表面活性剂对酶构象的影响。在阴离子型表面活性剂(SDS、SDSo)存在时,BLC发生了三级结构由松弛到展开的渐变过程,α-螺旋和β-折叠的分数也同时发生变化,无规则卷曲分数增加,直至酶分子完全展开。在含SB3-12的体系中,酶的二级和三级结构没有发生明显的变化。DTAB使酶分子的三级结构发生了明显的变化,但是由于酶聚集体的生成,CD光谱不能检测酶的二级结构。在酶和SB3-12共存的溶液里加入SDS,在SDS过量以后,酶的二级和三级结构的变化与没有SB3-12时有类似的规律。结合这些酶活性、分子间相互作用热力学及酶结构的研究结果,发现各种性质的变化与表面活性剂的浓度相关,据此提出了所研究的表面活性剂与BLC相互作用的机理。4.本课题组前期工作研究表明α-糜蛋白酶(α-CT)在阳离子型Gemini表面活性剂存在下具有超活性。作为此研究工作的延伸进一步研究了12-10-12与α-CT混合体系。通过等温滴定量热法系统地测定了在12-10-12浓度小于cmc时与α-CT的相互作用焓,讨论了在12-10-12缓冲溶液中酶的超活性及低的稳定性的原因。
[Abstract]:Compared with many synthetic catalysts, enzymatic catalysis has the advantages of high selectivity and catalytic efficiency, less toxicity and mild reaction conditions, and is essential for various biochemical processes in organisms. Mechanical stability. Surfactants are additives frequently used in enzyme catalytic reaction systems, which can regulate the catalytic activity and stability of enzymes. In this paper, anionic, cationic and amphoteric surfactants and catalase (BLC) were studied by means of isothermal titration calorimetry (ITC), fluorescence spectroscopy, circular dichroism and ultraviolet-visible spectroscopy. The main contents and results are as follows: 1. The effects of surfactants on enzyme activity were characterized by UV-vis spectroscopy. The results showed that anionic surfactants (SDS, SDS) had significant effects on enzyme activity. O) inhibited the activity of BLC at low concentrations, and denatured the enzyme near their respective critical micelle concentrations (cmc). When cationic surfactants (DTAB) were gradually added to the BLC solution, catalase undergone a process of aggregation and redispersion before the concentration of DTAB reached cmc, and the relative activity of catalase decreased to about 80%. Amphoteric betaine surfactant (SB3-12) has no obvious effect on the activity of the enzyme and has a certain protective effect on the natural structure of the enzyme. In the presence of SB3-12, excessive SB3-12 can inhibit the effect of SDS on the activity of BLC. It is found that when xSB3-120.5, BLC can still maintain high activity, while x SB3-12 exists. The activity of BLC was controlled by the molar fraction of two surfactants. When BLC was cultured in the mixed surfactant solution of DTAB and SB3-12, the activity of BLC was controlled by the concentration of DTAB. 2. The interaction between surfactants and enzyme molecules was studied by isothermal titration calorimetry. Thermodynamics of the interaction between sex agents and enzymes is related to the charge properties of the polar head groups. Ionic surfactants first bind to the opposite charge sites on the enzyme molecules. The electrostatic interaction between SDS and enzymes is accompanied by hydrophobic interaction between alkyl chains and enzymes. Excess SDS forms molecular clusters on the surface of enzymes, and enzymes unfold when DTAB and DTAB interact with each other. Due to the absence of hydrophobic domains near the negative charge sites on the enzyme surface, excessive DTAB clusters on different enzyme molecules can aggregate into shared micelles, which promotes the proximity and precipitation of BLC molecules. The interaction between SB3-12 and enzyme molecules is weaker than that between DTAB or SDS and enzyme molecules. The interaction between SB3-12 and enzyme molecules is controlled by their molar fraction. When xSDS is 0.50, SDS molecules cooperate with enzyme molecules. When xSDS is 0.50, excessive SDS acts on the BLC molecule to expand the enzyme molecule. For SB3-12/DTAB/BLC mixtures, SB3-12 does not affect the electrostatic interaction between DTAB and BLC, but can promote the denaturation of BLC molecule. Mixed or shared micelles of SB3-12 and DTAB were formed to reduce the concentration of DTAB which agglomerated the BLC. 3. The effects of different surfactants on the conformation of the enzyme were studied by fluorescence spectroscopy and circular dichroism spectroscopy. In the system containing SB3-12, the secondary and tertiary structures of the enzyme did not change significantly. DTAB changed the tertiary structure of the enzyme molecule significantly, but CD spectra did not change because of the formation of enzyme aggregates. It can be used to detect the secondary structure of enzymes. Adding SDS into the coexistence solution of enzymes and SB3-12, the changes of secondary and tertiary structure of enzymes are similar to those without SB3-12. Combining these enzymes, the thermodynamics of intermolecular interaction and the structure of enzymes, the changes of various properties and the concentration of surfactants are found. The mechanism of the interaction between the surfactant and BLC was proposed. 4. Previous work of our group showed that alpha-chymotrypsin (alpha-CT) had superactivity in the presence of cationic Gemini surfactant. As an extension of this work, the mixed system of 12-10-12 and alpha-CT was further studied. The enthalpy of interaction with alpha-CT at 12-10-12 concentration less than CMC was measured systematically. The reasons for the superactivity and low stability of the enzyme in 12-10-12 buffer solution were discussed.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.8
本文编号：2225861
[Abstract]:Compared with many synthetic catalysts, enzymatic catalysis has the advantages of high selectivity and catalytic efficiency, less toxicity and mild reaction conditions, and is essential for various biochemical processes in organisms. Mechanical stability. Surfactants are additives frequently used in enzyme catalytic reaction systems, which can regulate the catalytic activity and stability of enzymes. In this paper, anionic, cationic and amphoteric surfactants and catalase (BLC) were studied by means of isothermal titration calorimetry (ITC), fluorescence spectroscopy, circular dichroism and ultraviolet-visible spectroscopy. The main contents and results are as follows: 1. The effects of surfactants on enzyme activity were characterized by UV-vis spectroscopy. The results showed that anionic surfactants (SDS, SDS) had significant effects on enzyme activity. O) inhibited the activity of BLC at low concentrations, and denatured the enzyme near their respective critical micelle concentrations (cmc). When cationic surfactants (DTAB) were gradually added to the BLC solution, catalase undergone a process of aggregation and redispersion before the concentration of DTAB reached cmc, and the relative activity of catalase decreased to about 80%. Amphoteric betaine surfactant (SB3-12) has no obvious effect on the activity of the enzyme and has a certain protective effect on the natural structure of the enzyme. In the presence of SB3-12, excessive SB3-12 can inhibit the effect of SDS on the activity of BLC. It is found that when xSB3-120.5, BLC can still maintain high activity, while x SB3-12 exists. The activity of BLC was controlled by the molar fraction of two surfactants. When BLC was cultured in the mixed surfactant solution of DTAB and SB3-12, the activity of BLC was controlled by the concentration of DTAB. 2. The interaction between surfactants and enzyme molecules was studied by isothermal titration calorimetry. Thermodynamics of the interaction between sex agents and enzymes is related to the charge properties of the polar head groups. Ionic surfactants first bind to the opposite charge sites on the enzyme molecules. The electrostatic interaction between SDS and enzymes is accompanied by hydrophobic interaction between alkyl chains and enzymes. Excess SDS forms molecular clusters on the surface of enzymes, and enzymes unfold when DTAB and DTAB interact with each other. Due to the absence of hydrophobic domains near the negative charge sites on the enzyme surface, excessive DTAB clusters on different enzyme molecules can aggregate into shared micelles, which promotes the proximity and precipitation of BLC molecules. The interaction between SB3-12 and enzyme molecules is weaker than that between DTAB or SDS and enzyme molecules. The interaction between SB3-12 and enzyme molecules is controlled by their molar fraction. When xSDS is 0.50, SDS molecules cooperate with enzyme molecules. When xSDS is 0.50, excessive SDS acts on the BLC molecule to expand the enzyme molecule. For SB3-12/DTAB/BLC mixtures, SB3-12 does not affect the electrostatic interaction between DTAB and BLC, but can promote the denaturation of BLC molecule. Mixed or shared micelles of SB3-12 and DTAB were formed to reduce the concentration of DTAB which agglomerated the BLC. 3. The effects of different surfactants on the conformation of the enzyme were studied by fluorescence spectroscopy and circular dichroism spectroscopy. In the system containing SB3-12, the secondary and tertiary structures of the enzyme did not change significantly. DTAB changed the tertiary structure of the enzyme molecule significantly, but CD spectra did not change because of the formation of enzyme aggregates. It can be used to detect the secondary structure of enzymes. Adding SDS into the coexistence solution of enzymes and SB3-12, the changes of secondary and tertiary structure of enzymes are similar to those without SB3-12. Combining these enzymes, the thermodynamics of intermolecular interaction and the structure of enzymes, the changes of various properties and the concentration of surfactants are found. The mechanism of the interaction between the surfactant and BLC was proposed. 4. Previous work of our group showed that alpha-chymotrypsin (alpha-CT) had superactivity in the presence of cationic Gemini surfactant. As an extension of this work, the mixed system of 12-10-12 and alpha-CT was further studied. The enthalpy of interaction with alpha-CT at 12-10-12 concentration less than CMC was measured systematically. The reasons for the superactivity and low stability of the enzyme in 12-10-12 buffer solution were discussed.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.8
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