Portal de Periódicos da CAPES

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

1999; Elsevier BV; Volume: 732; Issue: 1 Linguagem: Inglês

10.1016/s0378-4347(99)00267-4

1872-812X

M. L. Miller, Gail V.W. Johnson,

Erythrocyte Function and Pathophysiology

Tissue transglutaminase (tTG) is a calcium-activated enzyme which can covalently crosslink the ϵ-amino group of a peptide-bound lysine into the γ-carboxamide group of a peptide-bound glutamine, forming a ϵ-(γ-glutamyl)lysine isopeptide bond. We have developed a sensitive, single-step method for the isolation and detection of tTG-mediated isopeptide bonds from purified proteins and tissue homogenates. This method offers significantly improved resolution over current techniques, and obviates the need for multi-column systems or costly fluorescence monitors. By using enzymatic proteolysis, derivatization with phenylisothiocyanate, and a simple elution 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 τ proteins (0.75 mol of isodipeptide per mol of τ), and whole-tissue liver homogenates (0.5 nmol of isodipeptide per mg of total protein).