Investigation On The Molecular Interaction Between Different Structure Types Of Alternaria Mycotoxins And Serum Albumin

Alternaria mycotoxins are widely contaminated in crops and foods with high exposure levels,but lack of in-depth toxicity and risk assessment studies,and lack of limit standard.Serum albumin–toxin binding study,as a widely employed risk assessment technique,can help us understand the toxicokinetics of toxins.In this paper,the binding interaction between different structure types of Alternaria toxins and model serum albumin Human serum albumin(HSA)and Bovine serum albumin(BSA)were investigated using steady-state fluorescence spectroscopy,synchronous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy,UV spectroscopy,Circular dichroism spectroscopy and molecular docking methods under the simulated blood physiological p H conditions(p H=7.4).And the effects of molecular structure and chemical properties of Alternaria toxins on the interaction process were discussed,in order to lay a solid foundation for studies related to in vivo exposure to Alternaria toxins.The main results are as follows:(1)For the first time,the interaction between the representative structural type of Alternaria toxins with HSA and BSA were systematically studied.The result showed that the dibenzopyrone structural toxins alternariol monomethylether(AME),alternariol(AOH)and the tetramic acid structural toxin tenuazonic acid(TeA)can all quench the fluorescence of serum albumin protein by static quenching mechanism.When interacting with serum albumin of different species models,the affinity of different structure types of Alternaria toxins showed certain differences.The binding constants of the tetramic acid structural toxin was higher than those of the dibenzopyrone structural toxins,and the binding constants of these toxins with BSA were higher than those with HSA.Especially,the binding constants between tetramic acid derivatives TeA(1.32×10~4-3.17×10~4L/mol)with HSA is stronger than that of dibenzopyrone derivatives AME and AOH(0.44-3.85×10~3L/mol),Under the condition of 37°C,the binding constants of the three Alternaria toxins with serum albumin is TeA>AME>AOH,and the binding constant of TeA with BSA was as high as 1.95×10~5L/mol.(2)Thermodynamic research results showed that the negativeΔG values of all interaction systems reveal that the binding interaction is spontaneous.The negativeΔH values andΔS values reveal that the dominant forces between AME/AOH and serum albumin are hydrogen bonds and van der Waals forces.The positiveΔH values andΔS values reveal that the dominant forces between TeA and serum albumin are hydrophobic forces.(3)According to the F(?)rster’s theory,the result shows that the energy transfer between serum albumins and the different structure types of Alternaria toxins with high probability.The binding distances between AME,AOH and TeA with HSA were 2.56,2.60 and 2.84 nm,the binding distances between AME,AOH and TeA with BSA were2.37,2.54 and 2.92 nm.The binding distance AME<AOH indicates that compared with AOH,the affinity of AME and serum albumin is stronger.The binding distance of the tetramic acid structural toxin TeA is greater than that of AME and AOH,but the binding constant of TeA is greater than that of the dibenzopyrone structural toxins AME and AOH.Therefore,we assume that the binding position of TeA on serum albumin may be different from AME and AOH.(4)The parameters of the different structure types of Alternaria toxins and serum albumin interaction indicate toxins has induced a slight conformational change in serum albumin structure.The increase in the hydrophobicity surrounding Trp residues caused by AME and AOH banding to serum albumin,and the degree of Trp residue fluorescence quenching is more obvious than Tyr residue fluorescence quenching.These results indicate that the binding site for AME and AOH is siteⅠ.The decrease in the hydrophobicity surrounding Trp residues caused by TeA was observed,and the degree of Trp residue fluorescence quenching and Tyr residue fluorescence quenching is basically the same,that indicate that the binding site for TeA is different from AME and AOH.The CD results also showed that the binding of the two structural types of Alternaria toxins had different effects on the structure of serum albumin.The binding of AME and AOH reduced the content ofα-helix structure in HSA from 48.93%to 39.41%and 44.01%,respectively,and reduced the content ofα-helix structure in BSA from30.68%to 28.21%and 27.26%,respectively.We assume that the binding of AME and AOH destroyed part of the hydrogen bond network that stabilizes the secondary structure of serum albumin.However,the binding of TeA increased theα-helix content in HSA from 48.93%to 52.76%,and increased theα-helix content in BSA from 30.68%to 35.62%.The hydroxyl group,ketone group and N atom,which is located on the pyrrole ring in TeA may formed hydrogen bonds with amino acid residues,thus,stabilizes the structure of serum albumin.(5)The competitive binding experiments and molecular docking showed that AME and AOH binds to sites I,and TeA binds to site II of HSA,verified the results in(3)and(4).And the affinity between the three Alternaria toxins and serum albumin is TeA>AME>AOH,which was consistent with the results of spectral studies.Molecular docking showed that AME formed hydrogen bonds with Arg218,Ala291 and Lys436,the methoxy moiety structure forms hydrophobic interactions with hydrophobic amino acids such as Tyr452 and Val455,the aromatic rings could formπ→πstacking interactions with Trp214 and Tyr452.AOH formed hydrogen bonds with Lys195 and Pro447,the methyl structure forms hydrophobic interactions with Val455,the aromatic rings could formπ→πstacking interactions with Trp214.TeA can form a hydrogen bond with Tyr411,and the alkyl part extends into the hydrophobic pocket,forming a strong hydrophobic interaction with hydrophobic amino acid residues such as Leu387,Leu453 and Leu407,and enhancing the affinity of TeA and HSA.