Protein L-Dopa as an index of hydroxyl radical attack on protein tyrosine

It is widely believed that hydroxyl radicals generated in vivo contribute 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 attack on proteins. The catechol structure of Dopa makes it amenable to isolation with alumina, followed by HPLC analysis, typically used for the measurement of catecholamines. Because a level of controversy exists about the formation of Dopa by hydroxyl radicals, we conducted a systematic study of the formation of Dopa from tyrosine, tyrosine dipeptides, pure proteins (chymotrypsin and myelin basic protein), and endogenous proteins in tissue homogenates (rat brain), exposed to hydroxylating conditions (Fe2+/EDTA/ascorbate at neutral pH). Dopa residues in peptides 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 hydrolysis conditions was prevented with added phenol; chymotrypsin and precipitated pellets of brain protein were also protective. Overall recoveries (hydrolysis plus purification procedures) averaged 83.4 +/- 1.7%. This improved analytic procedure may be useful far studying protein damage by hydroxyl radicals. (C) 1998 Academic Press.