Study On The Interaction Of Three α-dicarbonyl Compounds With HSA And DNA And The Inhibition Of Their Glycosylation

As a highly active glycosylation intermediate,α-Dicarbonyl compounds（α-DCs）would further cross link glycosylation with proteins.The misfolding and aggregation of glycosylated proteins would lead to conformational changes,form amyloid fibers which affects the biochemical function of proteins,and lead to the formation of advanced glycation end products（AGEs）,which will further lead to diabetes and its complications,neurodegenerative diseases,metabolic syndrome and aging.AGEs andα-DCs have been reported to have an impact on biological macromolecular:DNA and proteins.The molecular mechanism of the interaction betweenα-DCs and biological macromolecules（including specific binding sites,binding forces and binding capabilities）is rarely studied.The most common harmful effect ofα-DCs is to induce protein glycosylation,and the influence of protein glycosylation on the human body could not be underestimated.Therefore,it is urgent to find effective inhibition means.In recent years,blocking protein glycosylation process by anti-glycosylation agents and inhibiting the formation of AGEs had become a research focus.Natural active products have become potential anti-glycosylation agents due to their excellent safety and high efficiency.In addition,reducing the entry of precursor substancesα-DCs and related harmful substances into the human body from the source is also one of the important ways to reduce glycation reaction.α-DCs in the human body mainly comes from food intake,such as Maillard reaction,caramelization or lipid oxidation occurs in sugar or fat rich food（honey,baked goods,roast meat,etc.）.Therefore,it is of positive significance to explore the inhibitory effect of natural active substances on the formation ofα-DCs and glycosylation-related hazards in food models.Phloretin and phlorizin have been proved that they could alleviate diabetes and inhibit glycosylation.However,there were few studies on the mechanism of inhibiting glycosylation of protein,and even less studies on inhibiting the formation of hazardous substances from Maillard reaction in food chemical models.Threeα-DCs,methylglyoxal（MGO）,2,3-butanedione（DA）and glyoxal（GO）,were used in this paper.Firstly,the interaction mechanisms of the threeα-DCs with calf thymus DNA（ct DNA）and human serum albumin（HSA）were investigated.The influence of glycosylation on protein structure of the threeα-DCs was explored and compared,and the relationship between the glycosylation ability and binding affinity of differentα-DCs was analyzed.In order to inhibit the damage ofα-DCs glycosylation on protein,the inhibitory activity and mechanism of dietary polyphenol phloretin and phlorizin on protein glycosylation model in vitro were investigated.In addition,a food chemical simulation system was established to explore the effects of phloretin and phlorizin on protecting substrates,inhibiting the formation of harmful substances and removing part of harmful substances in Maillard reaction,and clarify their inhibitory mechanism in Maillard fever reaction.The main research contents and results are as follows:1.The interaction between MGO/DA/GO and ct DNA was determined by multi-spectral method combined with computer simulation.UV spectrum analysis showed that van der Waals forces and hydrogen bonds drove the spontaneous binding of MGO/DA/GO with ct DNA.The energy distribution in molecular dynamics simulations supported this conclusion.At 298 K,the binding constants of MGO/DA/GO with ct DNA were close to those of classical fluted binder,with the values of 2.99×10³,1.96×10³and 1.05×10³L mol^-1,respectively.The results of Na Cl,single and double stranded DNA,thermal denatability,viscosity and circular dichroic chromatography demonstrated that MGO/DA/GO and ct DNA were combined by groove mode.Molecular docking visualized MGO/DA/GO binding in AT-rich sulci regions of DNA,where DT7 and DA18 were the binding active sites.Molecular dynamics simulation showed that MGO-ct DNA complex had higher root-mean-square deviation（RMSD）,cyclotron radius（Rg）and root-mean-square fluctuation（RMSF）values than free ct DNA,indicating that the binding of MGO made the DNA structure partially loose and the stability decreased.Gel electrophoresis experiments showed that MGO/DA/GO could damage plasmid DNA in the presence of lysine/Cu²⁺,and MGO and GO could even completely destroy the superhelix morphology of DNA.2.Multispectral analysis and molecular docking showed that MGO/DA/GO were bound to theⅡA subdomain（siteⅠ）of HSA mainly by hydrogen bonding and van der Waals forces.Molecular dynamics studies showed that MGO mainly affected Lys and Arg residues of HSA,resulting in a more stable skeleton and sparse structure of HSA.The glycosylation process of HSA induced by MGO/DA/GO was studied.The results showed that the glycosylation ofα-DCs enhanced the levels of browning,fluorescence AGEs and the oxidation of proteins.The structure of glycosylated HSA had obvious changes,including the increase of carbonyl content,the decrease of amino,sulfhydryl,surface hydrophobicity andα-helix content,and the formation of cross-linked structural proteins andβ-amyloid proteins.The glycosylation intensity and binding affinity of the threeα-DCs showed a consistent order:MGO>DA>GO,indicating that the binding affinity of MGO/DA/GO with HSA may be the internal mechanism of the different ability of MGO/DA/GO to induce HSA glycosylation.3.In MGO-HSA glycosylation system,both of phloretin and phlorizin could protect free amino and sulfhydryl groups from damage,and reduce the content of protein carbonyl and oxidation products,so as to maintain the structural stability of HSA during the glycosylation process.Phloretin and phlorizin inhibited the formation of fluorescent AGEs,amyloid cross-linked structures and protein aggregates.The results of LC-Qq Q-MS showed that phloretin and MGO could form single MGO-phloretin and double MGO-phloretin adducts to remove MGO,while phlorizin first removed sugar molecules to form phloretin and then played a trapping role.The scavenging rates of MGO were 6.44 L mmol^-1h^-1and 0.79 L mmol^-1h^-1in phloretin and phlorizin,respectively(AG was 0.33 L mmol^-1h^-1in positive control).Phloretin and phlorizin also showed strong activity in clearing O₂^-·and·OH in Lys-MGO system,chelating Fe²⁺and reducing Fe³⁺.By protecting the structure of HSA in the glycosylation process,reducing the biogenic conjugate products,trappingα-DCs,clearing free radicals produced in the glycosylation system,and acting on catalytic metal ions,it may be the anti-glycosylation mechanism of phloretin and phlorizin.Their inhibitory capacity at low experimental concentrations（30～240μM）was as follows:phloretin>phlorizin>AG.4.The chemical simulation system of Maillard（Lys-Fru）and monosaccharide decomposition（Fru）was established.After high temperature heat treatment,the systems were browning and p H value decreased in the system,Fru consumption was higher than that of Lys,and a variety of harmful products were detected.Phloretin and phlorizin had no significant effect on the browning and p H change of the system,but they could slow down the consumption of reaction substrates（Lys and Fru）.In addition,the contents ofα-DCs（MGO,DA and GO）,5-HMF,AA,imidazoles and AGEs were significantly reduced in Lys-Fru system containing phloretin and phlorizin.MGO,DA,GO and 5-HMF could also be produced by individual Fru after heating,and their formation was also inhibited by phloretin and phlorizin.Overall,the inhibitory activity of phloretin was superior to phlorizin.These results indicated that,on the one hand,phloretin and phlorizin obstructed the participation of substrates in the decomposition of Maillard and monosaccharides,and on the other hand,inhibited the production and removal of a variety of harmful substances during the thermal reaction,so as to achieve the inhibitory effect.