The Formation, Isolation and Importance of Isopeptides in Heated Proteins

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Abstract
The separation and resolution of the isopeptides Nε(γ-Lglutamyl)-L-lysine and Nε(β-aspartyl)-L-lysine, formed in heated proteins, has been successfully achieved. The method demands a well characterised ion-exchange column and the use of pH 3.40 lithium citrate buffer (O.2N Li+). Due to variations in particle size and percentage crosslinkages in the ion-exchange resin a computer assisted buffer gradient system has been developed. This system affects resolution of both isopeptides in 7h. The use of leucyl-glycine as an internal standard facilitates quantitative estimation of the isopeptides. This separative method has been used to analyse a series of heated protein samples and to estimate the quantities of isopeptides formed. The ability of a protein to form isopeptide links is discussed as well as the implication of such links on the reactivity and digestibility of proteins.