Sp. Gieseg et al., PROTEIN-BOUND 3,4-DIHYDROXYPHENYLALANINE IS A MAJOR REDUCTANT FORMED DURING HYDROXYL RADICAL DAMAGE TO PROTEINS, Biochemistry, 32(18), 1993, pp. 4780-4786
Proteins and aromatic amino acids previously exposed to hydroxyl radic
als reduced cytochrome c, free iron, and copper ions. A major product
of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine
(DOPA), which has these reducing properties. The reduction of nitro b
lue tetrazolium by radical-damaged protein was consistent with the gen
eration of quinones in the protein. By acid hydrolysis followed by hig
h-performance C18 reversed-phase liquid chromatography we have shown t
hat hydroxyl radical-damaged proteins contain significant amounts of p
rotein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was
confirmed by gas chromatography-mass spectrometry. PB-DOPA was also g
enerated enzymatically using mushroom tyrosinase, which catalyzes the
hydroxylation of tyrosine residues. By comparing the levels of DOPA in
radical-damaged or enzyme-treated protein with that of cytochrome c r
eduction, we show that PB-DOPA is a major source of the observed reduc
ing activity. PB-DOPA may have a role in the replenishment of reduced
transition metal ions involved in free radical generating systems in v
ivo.