A SENSITIVE FLUOROMETRIC ASSAY FOR PROTEIN-BOUND DOPA AND RELATED PRODUCTS OF RADICAL-MEDIATED PROTEIN OXIDATION

Oxidative attack on proteins results in the hydroxylation of tyrosyl r esidues to protein-bound DOPA (3,4-dihydroxyphenylalanine). Existing m ethods for assaying protein-bound DOPA have poor sensitivity and numer ous possible interferences, such that accurate determination (especial ly of very low DOPA concentrations) has required time-consuming acid h ydrolysis and HPLC analysis with fluorometric detection. This work pre sents a sensitive and selective assay for peptide or protein-bound o-b enzoquiuones derived from DOPA based on fluorometric detection of ethy lenediamine derivatives. Detection limits for protein-bound DOPA are i n the range 0.53-4.70 ng/mL for the assay mixture, corresponding to sa mple DOPA concentrations of 0.59-5.30 ng/mL (representing a minimum of 6-54 pmole detected), depending on the particular protein/peptide und er study. The assay response increases linearly with DOPA concentratio n, and also with the extent of radical exposure of the protein. The as say is a simple and fast way to assess DOPA formation and thus oxidati ve damage in a protein.