Rapid, single-step procedure for the identification of transglutaminase-mediated isopeptide crosslinks in amino acid digests

Tissue transglutaminase (tTG) is a calcium-activated enzyme which can coval ently crosslink the epsilon-amino group of a peptide-bound lysine into the gamma-carboxamide group of a peptide-bound glutamine, forming a epsilon-(ga mma-glutamyl)lysine isopeptide bond. We have developed a sensitive, single- step method for the isolation and detection of tTG-mediated isopeptide bond s from purified proteins and tissue homogenates. This method offers signifi cantly improved resolution over current techniques, and obviates the need f or multi-column systems or costly fluorescence monitors. By using enzymatic proteolysis, derivatization with phenylisothiocyanate, and a simple elutio n gradient for HPLC, we were able to determine the frequency of crosslinks in purified fibrin (1.7 mol of isodipeptide per mol of fibrin), crosslinked tau proteins (0.75 mol of isodipeptide per mol of tau), and whole-tissue l iver homogenates (0.5 nmol of isodipeptide per mg of total protein). (C) 19 99 Elsevier Science B.V. All rights reserved.