Isolation,Purification,Identification And Characterization Of 2S Albumin Like Protein From Paeonia Suffruticosa

2S albumin proteins are seed storage proteins widely distributed in plant seeds which are with low molecular weight.It contains conserved motif of eight cysteine residues,which can form two intermolecular disulfide bonds and two intramolecular disulfide bonds,making these proteins very stable and compact,and these proteins have good water solubility and thermal stability.The members of this family have been bestowed with one or more functions which may include antimicrobial,anticancer activitivities,serine proteinase inhibitory activities and plant defense.Paeonia suffruticosa Andr is a special ornamental and medicinal shrub of Paeonia in China,which has the functions of antioxidant,antibacterial,anti-inflammatory,anticoagulant and hypoglycemic.The peony seed meal is the by-product of oil extraction,which is rich in functional substances and its protein content is about 30%.Studying functional protein of peony seed meal can not only enrich the basic research of peony seed protein,but also improve the added value of the seed meal and extend the industrial chain of peony seed.In this study,a new 2S albumin like protein（Ps-2S-Alb）was isolated and purified from PSMP.It was identified by mass spectrometry and de novo sequencing.Its structure was analyzed by bioinformatics,and its antioxidant activity was studied.The details are as follows:1. Extraction of water-soluble protein from peony seed meal.The water-soluble protein from defatted peony seed meal was extracted by water.Effects of solvent concentration,solvent and solute ratio（S/S）,and homogenate time on the yield of protein was studied.Based on the single factor experiment,three-factor and three-level response surface method was adopted to optimize the system.The optimum conditions were as follows:the concentration of extraction solution was 0.015 mol/L,the solvent and solute ratio was 1:8 and the homogenate time was 120 s.The yield of protein was 38.57±0.79%under the optimum conditions.2. Isolation and purification of Ps-2S-Alb.The water-soluble protein of peony seed meal was salted out by saturated ammonium sulfate,and then the protein component with 40-60%ammonium sulfate saturation was further purified by anion exchange chromatography column.And the elution conditions of anion exchange chromatography were optimized.The target components were collected and analyzed by SDS-PAGE after dialysis and desalting.The results showed that the molecular weight of the target protein was about 12 k Da,and the purity was over 95%.3. Identification of Ps-2S-Alb.The molecular weight of Ps-2S-Alb was 12.75 k Da by MALDI-TOF-MS,which was consistent with SDS-PAGE.The isoelectric point of Ps-2S-Alb was 5.8.The PAS staining gel electrophoresis revealed that the Ps-2S-Alb was glycoprotein and the glycosidic bond type was O-linkage glycosidic bond.LC-MS/MS combined with database searching was used for mass spectrometry identification.The results showed that no matching results with the target protein were found,indicating that the target protein may be a new protein that has not been reported.Therefore,in order to further identify,we used de novo sequencing to determine the full sequence of the target protein.Through blast analysis in the database,we found that it has high sequence homology with 2S albumin protein family,and named it Ps-2S-Alb.4. Study on the conformation of Ps-2S-Alb.DSC was used to study the thermal stability of Ps-2S-Alb,and the results show that the denaturation temperature of Ps-2S-Alb is 79.01°C,and the enthalpy value is 16.04 J/g.The secondary structure of Ps-2S-Alb was determined by CD,and the results show that Ps-2S-Alb is mainlyα-helix.The effects of different conditions（containing p H,temperature,denaturant）on the conformation of Ps-2S-Alb were investigated.The results showed that the conformation of Ps-2S-Alb was stable between p H 2.0～9.0,the stable structure of Ps-2S-Alb could be maintained at 30～70°C,the influence of four denaturants on the conformation of Ps-2S-Alb is DTT>SDS>Urea>Gdn HCl under the same conditions.5. Bioinformatics analysis of Ps-2S-Alb.The structure of Ps-2S-Alb was predicted by protein library and related servers.Bioinformatics analysis showed that Ps-2S-Alb predicted molecular weight of 12794 Da,isoelectric point value of 5.64,and molecular formula of C₅₂₈H₈₇₂N₁₇₄O₁₇₄S₁₁.The Ps-2S-Alb was predicted to be a stable hydrophilic proteinis,does not have a transmembrane region,does not belong to membrane protein,the secondary structure is mainly composed ofα-helix,and the Ps-2S-Alb contains four disulfide bonds.According to the predicted tertiary structure,Ps-2S-Alb is similar to the tertiary structure of 2S albumin from Brazil nut（c2lf A）in PDB database.6. Study on the antioxidant activity of Ps-2S-Alb.The antioxidant activity of Ps-2S-Alb was studied through the ability of reducing Fe³⁺,and scavenging ABTS,hydroxyl and DPPH free radicals in vitro.The results showed that Ps-2S-Alb could effectively scavenge DPPH,hydroxyl and ABTS radicals and had significant reduction ability to Fe³⁺in the concentration range of 0.1～0.5 mg/m L.The scavenging capacity of hydroxyl radicals was significantly stronger than that of ascorbic acid in the same concentration.