The Interaction Mechanism Between The Four Major Aflatoxins And Human Albumin Serum

Aflatoxins pollution is one of the world wide problems, particularly in developing countries. Aflatoxin B1(AFB1) is one of the strongest carcinogens, which enters the human food chain through a variety of ways directly or indirectly to contaminate grain and other agricultural products, food and feed. It was estimated that about 50 million people had been exposed to high levels of AFB1.Human serum albumin is the most abundant protein in blood plasma, which can bind, transport many endogenous and exogenous compounds and form non-covalent complex with exogenous in specific site. Human serum albumin is the pattern protein used to study the interaction between exogenous small molecules and biological macromolecules. Through studying the interaction, we can find the absorption, transport and metabolic processes of small molecules in blood.When toxins enter the body, they are transported to various organs and tissues through the HSA before metabolized. But the interaction between the toxin molecules and HSA had not been widely attented, especially mycotoxin molecules. According to the interaction between small drugs molecules and human serum albumin, it was speculated how toxins free concentration in vivo were affected by them binding to HSA. In fact, only free matter can penetrate cell membranes. Free concentration is related not only with toxin molecules total concentration and the concentration of HSA, but also the affinity of toxin molecules to HSA. Therefore, studying the binding interaction between toxins and HSA can effectively evaluate the biological effectiveness and precisely assessment of its toxicity. Furthermore, the reserach can play an guiding role in reducing the toxicity of AFs.In this paper, the interations between four major aflatoxins and HSA were investigated by fluorescence spectroscopy, UV absorption spectra, Synchronous fluorescence,circular dichroism, 3D fluorescence spectrum and molecular docking under the simulative physiological conditions(pH=7.4, Ionic strength 0.1). By studying, we got the binding mechanism,affinty constants, binding sites, binging distance, the major force, the change of serum albumin and so on. The interaction discussed on the molecular level, it could provide the action of aflatoxins metabolism in the boold. By studying the interactions of four different structure aflatoxins with HSA, the effect of different structures on the action processes was obtained.The main results were presented:1. The result showed that the four aflatoxins had a strong fluorescence quenching to HSA by fluorescence spectroscopy. Based on the Stern-Volmer equation, the main quenching mechanism between AFs and HSA was a static quenching process with forming ground state complex. According to the logarithmic equation, all binding sites(n) were approximate to 1 and the magnitude of binding constants were 104 at four different temperatures(298, 303, 308 and 313K), meaning one HSA molecule binding one toxin molecul and human serum albumin with a high affinity to AFs. At the same temperature,the order of four toxins quenching constant and affinity constant was AFB1> AFG1> AFB2> AFG2.2. The binding distance r between the AFB1, AFG1, AFB1, AFG2 and HSA were calculated to be 3.11 nm, 3.26 nm, 3.77 nm, 3.84 nm based on the theory of F?rster’s non-radiation energy transfer,illustrating that non-radiative energy transfer was one of the reasons for fluorescence quenching. The order of distance r between aflatoxins and HSA tryptophan was AFB1<AFG1 <AFB2 <AFG2, which indicated AFB1 was stored and transported by HSA most easily.3. According to Van’t Hoff equation, the negative enthalpy change(△H) and postive entropy change(△S) values indicated the four interactions were exothermic reactions and the negative Gibbs free energy(△G) showed they were spontaneous. The main forces between aflatoxins and HSA were hydrogen bonding and hydrophobic. The order of △G between aflatoxins and HSA was AFB1<AFG1<AFB2<AFG1, which indicated AFB1- HSA was most stable and the reaction was most likely to occur.4. By studying the effect of metal ions on AFs-HSA reaction, Zn2+, Mn2+ and Cu2+ could greatly reduce the affinity of HSA to AFs, indicating that they had a certain potential to prevent the effect of AFs on the lasting harm to people. while Fe3+ could increase the affinity of AFs to HSA, which was helpful for the sustained release of AFs molecules and increased the cancer risk after the individual intake of aflatoxins.5. The microenvironment of tryptophan residues increased hydrophobicity leaded by the four aflatoxins and HSA binding processes, which showed the environment of IIA hydrophobic cavity changed where Tryptophan located in. The ineractions also could change the HSA secondary structure, such as polypeptide chains C = O bond and α- helix content. The CD realved that HSA contained of 49.27% α-helix, and AFB1, AFB2, AFG1, AFG2 increased α- helix to 61.07%, 54.82%, 59.71%, 52.81% respectively. The order of HSA secondary structure changing degree was AFB1> AFG1> AFB2> AFG2.6. Analyzing the parameters of four aflatoxins and HSA reaction, the results showed that the aflatoxins were more poisonous,the greater degree of quenching HSA, the stronger the affinity to HSA, the greater degree of structural damaged, which can provided useful guidance for the establishment of evaluation criteria of aflatoxin toxicity based on HSA.7. The four aflatoxins binded in IB hydrophobic cavity of HSA by molecular docking method. Aflatoxins could formed hydrogen bonds with Arg-114, His-146, Tyr-138 of HSA. Due to the double bond, B1 and G1 could form π-π conjugation with His-146, Phe-146 and Phe-157, which made AFB1-HSA and AFG1-HSA more stable. The five-membered rings carbonyl of B1 and G1 made hydrogen bond length shorter, lending AFB1-HSA and AFG1-HSA more stable than AFB2-HSA and AFG2-HSA. This result consistented with the order of binding constants and the Gibbs free energy obtained.