The formation, isolation and importance of isopeptides in heated proteins

The separation and resolution of the isopeptides Nepsilon (gamma-L-glutamyl)-L-lysine and Nepsilon (beta-aspertyl)-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 isopeptides links is discussed as well as the implication of such links on the reactivity and digestibility of proteins.