PROTEIN L-DOPA AS AN INDEX OF HYDROXYL RADICAL ATTACK ON PROTEIN-TYROSINE

It is widely believed that hydroxyl radicals generated in vivo contrib ute to damage to macromolecules, such as proteins and DNA We evaluated methodology based on the transformation of protein tyrosine to L-Dopa , via aromatic ring hydroxylation, as an index of hydroxyl radical att ack on proteins. The catechol structure of Dopa makes it amenable to i solation with alumina, followed by HPLC analysis, typically used for t he measurement of catecholamines. Because a level of controversy exist s about the formation of Dopa by hydroxyl radicals, we conducted a sys tematic study of the formation of Dopa from tyrosine, tyrosine dipepti des, pure proteins (chymotrypsin and myelin basic protein), and endoge nous proteins in tissue homogenates (rat brain), exposed to hydroxylat ing conditions (Fe2+/EDTA/ascorbate at neutral pH). Dopa residues in p eptides and proteins were liberated by acid hydrolysis with 6 M HCI at 145 degrees C for 1 h. A marked lability of Dopa in 6 M HCI under hyd rolysis conditions was prevented with added phenol; chymotrypsin and p recipitated pellets of brain protein were also protective. Overall rec overies (hydrolysis plus purification procedures) averaged 83.4 +/- 1. 7%. This improved analytic procedure may be useful far studying protei n damage by hydroxyl radicals. (C) 1998 Academic Press.