Protein tyrosine nitration induced by heme/hydrogen peroxide: Inhibitory effect of hydroxycinnamoyl conjugates

Abstract The present study was designed to optimize the experimental conditions that govern the heme-catalyzed nitration of protein tyrosine residues by nitrite, and, within this framework, to study the effects of 3,5-dicaffeoylquinic acid and its methyl ester, both of which have been previously reported to be antioxidants and inhibitors of leukocyte functions. Although the presence of hydrogen peroxide is essential in cell-free systems, an excess of this compound was found to be detrimental, so much so that an increase in hemin concentration actually resulted in an inverse effect on the reaction, depending on the levels of fixed hydrogen peroxide. Unlike previous reports on nitrite-induced albumin tyrosine nitration, the optimal pH here was found to be 7.0. The two caffeoyl conjugates tested were found to be effective inhibitors of protein nitration, with IC 50 values ranging from 20 - 30 μM, regardless of the presence of bicarbonate. For the inhibition of myeloperoxidase-catalyzed protein nitration by human polymorphonuclear leukocytes stimulated by phorbol ester, the potencies obtained were up to two times higher. This is the first time that caffeoylquinic esters have been reported as inhibitors of heme-based protein nitration.