Mechanism of the Formation and Proteolytic Release of H2O2-induced Dityrosine and Tyrosine Oxidation Products in Hemoglobin and Red Blood Cells

Abstract

Oxyhemoglobin exposed to a continuous flux of H 2 O 2 underwent oxidative modifications, including limited release of fluorescent fragmentation products. The main fragments formed were identified as oxidation products of tyrosine, including dopamine, dopamine quinone, and dihydroxyindol. Further release of these oxidation products plus dityrosine was only seen after proteolytic degradation of the oxidatively modified hemoprotein. A possible mechanism is proposed to explain the formation of these oxidation products that includes cyclization, decarboxylation, and further oxidation of the intermediates. Release of dityrosine is proposed as a useful technique for evaluating selective proteolysis after an oxidative stress, because dityrosine is metabolically stable, and it is only released after enzymatic hydrolysis of the oxidatively modified protein. The measurement can be accomplished by high performance liquid chromatography with fluorescence detection or by high efficiency thin layer chromatography. Comparable results, in terms of dityrosine release, were obtained using red blood cells of different sources after exposing them to a flux of H 2 O 2 . Furthermore, dityrosine has been reported to occur in a wide variety of oxidatively modified proteins. These observations suggest that dityrosine formation and release can be used as a highly specific marker for protein oxidation and selective proteolysis.

Reaction of Human Hemoglobin with Peroxynitrite

“…Finally, it is possible that the oxo-ferryl complex oxidizes amino acid residues, in which case globin-derived radicals should be detected. This latter possibility is in line with previous results obtained with hydrogen peroxide (10). Thus, the mechanisms by which 1) oxyHb is oxidized, 2) ferrylHb is formed, and 3) ferrylHb is reduced back to metHb still remain to be defined.…”

“…Importantly, oxidation of oxymyoglobin by ⅐ NO also yields an intermediate complex, which promotes the oxidation of ethylene-1,2-13 C as detected by NMR (13); this result indicates that a hydroxyl radical-like species arises at low yields from the iron-catalyzed homolysis of complexed peroxynitrite and that it can escape the solvent cage to initiate free radical chemistry (13). In addition, superoxide anion released in the first step of the reaction (Equation 8) would spontaneously dismutate to O 2 and H 2 O 2 (Equation 12), which would initiate secondary oxidation reactions in oxy-or metHb to ferrylHb (10,17,18) and promote consumption of pre-existing nitrite in peroxynitrite preparations (Table I), which is facilitated by autocatalytic mechanisms (16) The oxidation of oxyHb by peroxynitrite yields a transient ferrylHb intermediate (8,9). This observation contrasts with the stability of the ferrylHb intermediate formed under excess of H 2 O 2 (10, 55), which can be observed even in the presence of nitrite (Fig.…”

“…This contrasts with the two-electron oxidation product obtained with oxyHb under excess of hydrogen peroxide (H 2 O 2 ), which yields a relatively stable ferrylHb intermediate (i.e. minutes) that can be observed directly by spectrophotometric techniques (10). If peroxynitrite oxidizes oxyHb by a two-electron oxidation process, nitrite and molecular oxygen should be produced in addition to ferrylHb (Equation Neither nitrite nor molecular oxygen yields have been assessed yet, so the mechanism proposed in Equation 1 cannot be confirmed with the available data.…”

“…The alternative scenarios for the peroxynitrite/oxyHb reaction are further complicated by the fact that the reactions of nitrite (NO 2 Ϫ ) and nitrogen dioxide ( ⅐ NO 2 ) with oxyHb could promote the formation of hydrogen peroxide (13,16) which in turn can secondarily react with oxy-and metHb to yield ferrylHb (Equation 3) or ferrylHb with a second oxidation equivalent centered in the globin moiety, respectively (10,17,18).…”

Identification of Free Radicals on Hemoglobin from its Self-peroxidation Using Mass Spectrometry and Immuno-spin Trapping

“…The globin radical-derived nitrone adduct can be investigated by immuno-spin trapping (16). In the absence of a spin trap, the globin-centered radical(s) can decay by formation of cross links (23), such as dityrosine (24) and possibly other cross-links.…”