Extraction And Structural Identification Of Yolk Glycoprotein In The Four Fish Roe

The fish roe is rich in yolk protein,which is composed of lipovitellin,phosphoprotein and ?-componet,all of which are glycoproteins.In recent years,there are many studies on physical and chemical properties and immunogenicity of fish yolk protein,but there are few researches on the structure of yolk protein.Glycans of glycoprotein are carrying lots of biological information,and analyzing of its structure can provide theoretical basis for studying glycoprotein biological activity and other features.In this study,Acipenser gueldenstaedti,Channaargus,Lateolabrax japonicas and Carassius auratus solution roe were experiment material,using two different kind of salt and extraction methods to extract yolk protein in four species of fish roe.The appropriate extraction method was determined finally by comparing the extraction rate of two methods and the electrophoresis distribution of the yolk protein.The specific protein in four species of fish roe was identified by protein spectrum analysis.The specific protein in Channaargus roe was selected for the structure analysis of part of glycans in glycoprotein.The results of this study are as follows:(1)Determination of yolk protein extraction in fish:Sodium chloride solution and Tris-buffer solution was used to extract yolk protein in the four roe,respectively.Comparing total protein and total sugar content with the two kinds of extract solutions,studies had shown that the extraction yield of these two kinds of solution were 38.1%and 25.7%,respectively,which was said that the extraction yield of sodium chloride solution was higher.SDS-PAGE analysis showed markedly different protein bands of four kinds of fish roe,all of which were clear and less extracted with sodium chloride solution.Among them,yolk protein bands in Channaargus and Lateolabrax japonicas roe were more pure.Channaargus roe had a high abundance and molecular weight of 26 kDa yolk protein,and there are two molecular weights of 14 kDa and 21 kDa yolk protein in Lateolabrax japonicas roe.There was no obvious change in the yolk protein strips extracted from Carassius auratus roe with two kinds of solutions.The yolk protein bands of Acipenser gueldenstaedti were significantly reduced.In order to facilitate the follow-up analysis,the fish yolk protein was extracted by sodium chloride solution.(2)Identification of specific proteins of P'-c:Selecting four different molecular weight proteins to analyze peptide fingerprint quality map identification,there were 18 kDa protein in Acipenser gueldenstaedti roe,26 kDa protein in Channaargus roe,14 kDa protein in Lateolabrax japonicas roe and 16 kDa protein in Carassius auratus roe,respectively.By LC-MS/MS analysis of the peptide segment identificated matched with Uniprot database,the results suggested that the sequence homologies were above 90%;Compared with vitellogenin sequence in Oncorhynchus keta(B7XGC3)and Gambusia affinis(Q589T0)known ?'-c sequence,the results showed they were in the P'-c domain and some fragments were aligned successly.The research suggested that the four species of yolk proteins were P'-c,all of which were in the vitellogenin VWFD domain.It was the first time to find four different molecular weight?'-c in four kinds of fish roes,and it also showed that ?'-c from different species was different.Compared multiple sequence alignment with the active peptides in the four species of fish roe,the result showed that the sequence homologies were low.Choosing nine different species of vitellogenin VWFD domain in database analyzed the multiple sequence alignment and amino acid composition.The results showed that there was a big difference in the different species of VWFD domain,namely further illustrating that ?'-c existed differences.These provided a theoretical basis for P'-c as a specific markedprotein.(3)Identification structure and coagulation activity of Channaargus yolk glycoprotein:The purity of the Channaargus protein was determined by SDS-PAGE electrophoresis separation and high performance liquid chromatography.The results showed that the purity of the Channaargus protein with sodium chloride extracted was 97%and very high.The extraction of Channaargus protein was glycoprotein by using the PAS staining method.The PNGase F enzyme and beta-elimination method were used to determine the sugar chain connection type of the Channaargus glycoproteins,and the results suggested that Channaargus glycoprotein contained N-glycoside and O-glycoside.In order to further study the structure of glycoproteins,gas chromatography was used to analyze of the sugar chain protein composition of Channaargus glycoprotein,and the results showed the monosaccharides composition of Channaargus glycoprotein were mannose:glucose:galactose = 1.2:1.0:12.2.Established the enrichment,purification and mass spectrometry identification method for N-linked glycans,the results showed that there was a quasi-molecular ion peak of 1580.34 m/z in Channaargus protein,and inferred that the N-linked glycan was made up of seven mannose and two acetylglucosamine(Man7GlcNAc2)through analysis of secondary fragmentary mass spectrogram,and the fracture mode of the secondary fragment of 1580.34 m/z was speculated.Finally,coagulation activity of the 26 kDa ?'-c protein of Channaargus was determined,the result showed that low concentration of Channaargus protein had high blood coagulation activity.