【作者】 李向荣；

【导师】 卢雁；

【作者基本信息】 河南师范大学 ， 物理化学， 2014， 博士

【摘要】 自由基是生物体内各种生化反应的中间体，由于存在未配对的电子，自由基具有很高的化学活性。正常情况下，自由基对生物体的生理调控必不可少。但是，当其产生过量或没有合理控制时，自由基就会表现出危险性，对脂质、核酸、蛋白质等生物大分子的结构和功能造成损伤。大量生物化学、生物学和临床医学研究表明，自由基对蛋白质造成的氧化损伤是许多慢性病的发病机理，也是导致衰老的主要原因。抗氧化剂可以提供氢原子或电子中和自由基的单电子。合理的补充抗氧化剂对维持身体健康、延缓衰老、预防或治疗疾病具有重要意义。自由基、抗氧化剂和蛋白质三者关系密切，自由基可以对蛋白质造成氧化损伤，而抗氧化剂可以清除自由基从而对蛋白质起到保护作用，同时抗氧化剂也可以与蛋白质相互作用导致蛋白质构象的变化。因此，研究抗氧化剂、自由基、蛋白质的相互作用机制十分重要。本文得到国家自然科学基金资助课题(No.21173071)和教育部高等学校博士点专项基金(No.20114104110002)资助，主要从以下四个方面开展研究工作：(1)抗氧化剂和自由基的相互作用；(2)自由基与蛋白质的相互作用；(3)抗氧化剂和血清白蛋白的相互作用；(4)抗氧化剂、自由基和蛋白质三元体系的相互作用。主要研究内容和结论如下：1.通过紫外-可见吸收光谱和荧光光谱测定了HSA/BSA清除DPPH(2,2-二苯代苦味酰肼基)自由基的能力以及它们之间的相互作用机制。结果表明HSA/BSA清除DPPH自由基的能力和谷胱甘肽类似。DPPH自由基与HSA/BSA的荧光猝灭机理为静态猝灭。DPPH自由基和HSA的结合存在两类不同的结合位点，结合数约为2，即一分子的HSA可以和两分子的DPPH自由基反应。DPPH自由基和BSA只有一类结合位点，结合数约等于1，即一分子的BSA只能和一分子的DPPH自由基反应。DPPH自由基与HSA/BSA的相互作用对色氨酸微环境有一定影响，但对酪氨酸微环境的影响不明显。2.采用紫外-可见吸收光谱研究了抗氧化剂(L-抗坏血酸、α-生育酚、β-胡萝卜素、虾青素、(+)-儿茶素、原花青素B3、谷胱甘肽、褪黑激素)清除DPPH自由基、羟基自由基以及超氧阴离子自由基的能力，其中原花青素B3清除DPPH自由基的能力最强，虾青素清除羟基自由基和超氧阴离子自由基的能力最强。3.在模拟生理条件下，通过等温滴定量热技术结合荧光光谱，紫外-可见吸收光谱，傅里叶红外光谱，圆二色谱以及分子模拟等方法，详细探讨了上述八种抗氧化剂与血清白蛋白的相互作用机制以及所引起的血清白蛋白构象的变化。研究结果表明不同的抗氧化剂与HSA/BSA的作用机制有所不同。L-抗坏血酸通过“非特异性”表面吸附结合到HSA/BSA的表面，结合位置位于HSA/BSA位置I和位置II的界面处，通过“作用域”猝灭HSA/BSA的内源荧光。α-生育酚与HSA/BSA为形成复合物的“特异性”结合，荧光猝灭机理为静态猝灭，结合在HSA/BSA位置II的疏水空腔内。β-胡萝卜素和虾青素与HSA/BSA的相互作用均为熵变和焓变协同驱动的自发过程，疏水和静电的综合作用是其主要的作用力类型，结合位置位于HSA/BSA的位置I和位置II的界面处，部分插入位置I的疏水空腔。(+)-儿茶素/原花青素B3与HSA的结合为有利的焓变和不利的熵变共同作用的结果，主要作用力是氢键和范德华力。(+)-儿茶素/原花青素B3与BSA的结合是焓变和熵变共同驱动所导致，静电和疏水作用是结合的主要作用力。(+)-儿茶素和原花青素引起HSA/BSA荧光猝灭机理均为形成基态复合物的静态猝灭，结合位置均位于HSA/BSA的位置I。谷胱甘肽通过两类互不干扰的结合位点吸附到HSA/BSA表面。褪黑激素与HSA/BSA形成1:1的复合物。虽然这八种抗氧化剂与HSA/BSA的作用机制不尽相同，但它们与HSA/BSA的结合常数均处于适中的范畴，利于其通过循环系统运输，并以合适的浓度到达各自的靶位置发挥药效。另外，这八种抗氧化剂与HSA/BSA的相互作用导致HSA/BSA二级结构中α-螺旋组分出现不同程度的降低，使血清白蛋白出现部分去折叠现象。4.通过荧光光谱研究了抗氧化剂、DPPH自由基和血清白蛋白三元体系的相互作用机制。研究表明三元体系的荧光猝灭机理为形成复合物的静态猝灭。结合常数和结合位点数与DPPH-HSA/BSA二元体系相比均有不同程度的降低，表明这八种抗氧化剂可以缓解DPPH自由基对血清白蛋白造成的氧化损伤，对血清白蛋白起到保护作用。更多还原

【Abstract】 Free radicals are necessary intermediates in a variety of normal biochemical reactions. A prominentfeature of radicals is that they have extremely high chemical reactivity, due to the presence of unpairedelectrons. Under normal conditions, free radicals are essential to the physiological control of livingorganisms. However, when generated in excess or not appropriately controlled, free radicals can causesevere damage to a broad range of macromolecules such as lipids, DNA and proteins. Abundantbiochemical, biological, and clinical evidence suggests the involvement of oxidative stress towards proteinsinduced by free radicals in the pathogenesis of various diseases and accelerated aging. Antioxidants canprovide hydrogen atoms or electrons to neutralize the single electron of radicals. Thus, this has attractedmuch attention to the role and beneficial effects of antioxidants in the maintenance of human health andprevention and treatment of diseases. In a word, free radicals, antioxidants and proteins are closely related.That is, free radicals can damage proteins. To protect against the harmful effects of radicals, antioxidantmolecules are of great importance. As well as, antioxidants can also bind to proteins. Therefore, it is verysignificant to explore the interaction mechanisms between antioxidants, free radicals and proteins.This work was supported by the National Natural Science Foundation of China (21173071) and theResearch Fund for the Doctoral Program of Higher Education of China (20114104110002). It mainlydiscussed by the following four aspects: The first part is about the interaction between antioxidants and freeradicals. The second part of this work focus on the studies of the interaction between free radicals andproteins. In a third part of the study, the interaction between antioxidants and proteins is studied bymicrocalorimetry and spectroscopy. At last, the interaction between the ternary system of antioxidants, freeradicals and proteins is studied. The major contents and conclusions are as follows:1. The abilities of HSA/BSA to scavenge DPPH radical were investigated using UV-vis absorptionspectra. It is shown that the antioxidant activity of HSA/BSA against DPPH radical is similar to glutathione.The interaction between HSA/BSA and DPPH was investigated using fluorescence spectroscopy. Theseresults indicate that DPPH quenches the fluorescence intensity of HSA/BSA through a static mechanism.The binding of DPPH to HSA exists two classes of binding sites with two different interaction behaviors. The value of the binding number approximately equals to2, suggesting that two molecules of DPPHradicals reduced by one molecule of HSA. BSA interacts with DPPH radical according to the independentbinding sites model and one molecule of DPPH radical reduced by one molecule of BSA. The binding ofDPPH to HSA/BSA has little effect on the microenvironment around Tyr residuesbut the effect is sufficientto perturb the environment in the vicinity of Trp residues.2. The abilities of these eight popular antioxidants (including L-ascorbic acid, α-tocopherol,β-carotene, astaxanthin,(+)-catechin, procyanidine B3, glutathione and melatonin) to scavenge several freeradicals such as DPPH radical, hydroxyl radical and superoxide anion radical were investigated usingUV-vis absorption spectra. It is shown that different antioxidants have the different abilities to scavengedifferent free radicals. This may be related to the different molecular structures of them. Procyanidine B3isthe best to eliminate DPPH radical, while, astaxanthin is the best to eliminate hydroxyl radical andsuperoxide anion radical.3. Under the physiological conditions, the interaction between these eight antioxidants and HSA/BSAwas investigated using isothermal titration calorimetry (ITC), in combination with fluorescencespectroscopy, UV-vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circulardichroism (CD) spectroscopy and molecular docking. The binding mechanism of antioxidants withHSA/BSA and the conformation changes of HSA/BSA were discussed. These results show that differentantioxidants interact with HSA/BSA through different binding mechanisms. The binding of L-ascorbic acidto HSA/BSA may be “nonspecific binding” by a surface adsorption mechanism that leads to coating of theprotein surface. The possible location of L-ascorbic acid is at the interface of site I and site II. Fluorescenceexperiments suggest that L-ascorbic acid is predominantly by the “sphere of action” quenching mechanism,whereas, for α-tocopherol, the quenching mechanism is “static quenching” and due to the formation of aground state complex. The binding of α-tocopherol to HSA/BSA is due to “specific binding” and thebinding site is mainly located within site II. The interaction between β-carotene/astaxanthin and HSA/BSAis synergistically driven by enthalpy and entropy, and the combined effect of hydrophobic forces andelectrostatic attraction play a major role in these reactions. The possible location of these two carotenoids isat the interface of site I and site II, and the molecule of β-carotene/astaxanthin partially inserts itself into ahydrophobic cavity of site I.(+)-Catechin/procyanidin B3binds to HSAis driven by favorable enthalpy and unfavorable entropy, and the major driving forces are hydrogen bond and van der Waals force. While, thebinding of (+)-catechin/procyanidin B3to BSA is synergistically driven by enthalpy and entropy and theelectrostatic interaction and hydrophobic interaction are the major binding forces in the binding of them.(+)-Catechin/procyanidin B3can quench the fluorescence of HSA/BSA through a static quenchingmechanism. Binding site I is found to be the primary binding site for them. The binding of glutathione toHSA/BSA is “nonspecific binding” by a surface adsorption mechanism, and exists two classes of bindingsites with two different interaction behaviors. The significant difference between glutathione andL-ascorbic acid is that the two different binding sites do not interfere with each other. For melatonin, onemolecule of HSA/BSA combines with one molecule of melatonin and forms a1:1melatonin-HSA/BSAcomplex. The obtained binding constants for these eight antioxidants with HSA/BSA are all in theintermediate range although they interact with HSA/BSA through different binding mechanisms. Themoderate affinity of these eight antioxidants for HSA/BSA leads to a faster diffusion rate in the circulatorysystem to reach target sites. These eight antioxidants indeed exert some influence on the conformation ofHSA/BSA. The decrease in α-helix structure of BSAand HSAsuggests a partial protein unfolding.4. The interaction mechanism between the ternary system of antioxidants, DPPH radical andHSA/BSA was investigated by fluorescence spectroscopy. The quenching mechanism of the ternary systemis “static quenching” and due to the formation of a ground state complex. The binding constants and thenumber of binding sites of the ternary system were decreased than that in DPPH-HSA/BSA binary system.These results show that these eight antioxidants can reduce oxidative damage induced by DPPH radical inserum albumins, playing a role in the protective effect.更多还原