| 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. |
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