Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins.
Proteins and aromatic amino acids previously exposed to hydroxyl radicals 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 blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated 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 reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.
Gieseg
S P
SP
Cell Biology Group, Heart Research Institute, Sydney, NSW, Australia.
Simpson
J A
JA
Charlton
T S
TS
Duncan
M W
MW
Dean
R T
RT
D002396:Catechols / Q000737:chemistry; D003300:Copper / Q000494:pharmacology; D003574:Cytochrome c Group / Q000737:chemistry; D004295:Dihydroxyphenylalanine / Q000737:chemistry*; D005609:Free Radicals; D007328:Insulin / Q000737:chemistry / Q000528:radiation effects; D008956:Models, Chemical; D014442:Monophenol Monooxygenase / Q000494:pharmacology; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry* / Q000187:drug effects / Q000528:radiation effects; D011809:Quinones / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000187:drug effects / Q000528:radiation effects; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000187:drug effects / Q000528:radiation effects
D002396:Catechols; D003574:Cytochrome c Group; D005609:Free Radicals; D007328:Insulin; D011506:Proteins; D011809:Quinones; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; D003300:Copper; D014442:Monophenol Monooxygenase