Study On Identification And Mechanism Of Xanthine Oxidase Inhibitory Peptides From Tuna Protein

Hyperuricemia refers to a series of diseases resulted by excess uric acid in blood,refluencing our life and health.Now,most clinic medicines(i.e.allopurinol)treating hyperuricemia have serious dependence and side-effect,which promotes the research of antihyperuricemic peptides for their high bio-activity and safty.Previous studies shown that tuna protein hydrolysate(TPH)can markedly reduce blood uric acid levels and inhibit xanthine oxidase(XO)activity in the liver and blood of hyperuricemic rats induced by injecting potassium oxonate.In order to reveal the sequences and XO inhibitory mechanisms of peptides in TPH,this article investigated the effect of different buffer on the XO inhibiton of TPH,enriched high XO inhibitory activity fraction from TPH by several separation technologies,identified the sequences of XO inhibitory peptides by MS/MS,synthesized those peptides and evaluated their XO inhibitory activity.In the last,XO inhibitory mechanism and structurefunction relationship of peptides from TPH were analized by combining evaluating data of XO inhibitory peptides and autodock technology to provide more professional insights to the research of XO inhibitory peptides and the production of uric acid lowing prptides.(1)The current method,determining the XO inhibition rate of peptides in vitro,directly refers to the determination method of XO inhibition of other small inhibitors without considering the influence of different buffer systems on the conformations of peptides and the enzyme.Therefore,the effects of three different buffer reagents on the XO inhibition rates of TPH were investigated.The results shown that the structure of TPH and XO had not significantly change in the three buffers.However,HEPES can slightly reduce the content of ?-helix and can be able to form a stronger interaction between XO and TPH comparing with other two buffers.All of these data are related to make TPH show a better XO inhibition in the HEPES system.This research provided a method to investigate the effect of buffers on the bioactivity of natural macromolecules.(2)Ethanol solution and Sephadex G-15 were used to separate the XO inhibitory peptides from TPH.The result demonstrated that the alcohol-soluble component(ESF)had the best XO inhibitory activity,which may be closely related to the high content of small molecule peptides(<1 kDa)and hydrophobic amino acid residues.There were eight components obtained from the separation by Sphadex G-15 and the P6 shown the best XO inhibitiory activity.Thirteen bi/tri-peptides were identified by UPLC-Q-TOF-MS/MS,wherein FH had the best XO inhibitory activity.Evaluating the XO inhibitory activity of those 13 peptides by comparing the HPLC method(used in this study)with the UV method(used in the previous report).This indicated that the UV method is not suitable for the determination of the peptides containing tryptophan residues(the maximum absorption peaks of tryptophan and urea are both at around 290 nm).(3)To further analyze the XO inhibitory mechanisms of the peptides,a molecular docking method was introduced in this study.Firstly,some laws of the action between popular XO inhibitor ligands and XO were summarized,which was used as an important criterion for judging the docking results between peptides and XO.The investigation of XO inhibitory mechanism found that FH can access into active centre of XO by comparing with xanthine(substrate)and interacts with Phe-914,Glu-802,Arg-880,and Thr-1010 residues.Comprehensive analysis of molecular docking results and XO inhibition of synthetic peptides can infer that Phe residue is vital for the peptides to show high XO inhibition.What's more,basic amino acids residues also show important contribution to the XO inhibitory activity of peptides.