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Glycinin, one of the predominant storage proteinsin soybeans, was examined as to whether it could be used as acalcium-binding mediator after chemical phosphorylation andenzymatic hydrolysis. Glycinin is composed of six subunits.One of the proglycinin subunits (A1aB1b) was overexpressed inE. coli to obtain nonphosphorylated proteins with homogeneity.To investigate the enhanced calcium-binding properties of thephosphopeptides, the proglycinin was purified, phosphorylated,and hydrolyzed with trypsin. The proglycinin expressed inE. coli was purified by ammonium sulfate precipitation, ionexchangechromatography, and cryoprecipitation. Chemicalphosphorylation by sodium trimetaphosphate was performedto obtain phosphorylated proglycinin. After the phosphorylation,one-dimensional isoelectric focusing gel electroanalysis confirmedthe phosphorylation of the proglycinin. The phosphorylatedpeptides were then hydrolyzed with trypsin, followed by abinding reaction with calcium chloride. The calcium-boundphosphopeptides were finally separated using immobilizedmetal (Ca2+) chromatography. Consequently, a limited tryptichydrolysate of the isolated phosphopeptides exhibited anenhanced calcium-binding ability, suggesting the potential ofglycinin phosphopeptides as a calcium-binding mediator withgreater availability.

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