Incorporation of the hydrophobic probe N-t-BOC-L-tyrosine tert-butyl ester to red blood cell membranes to study peroxynitrite-dependent reactions.

We have previously demonstrated that red blood cells (RBC) are an important sink of intravascularly generated peroxynitrite even in the presence of physiological concentrations of CO2 or other plasmatic biotargets. Once inside erythrocytes, peroxynitrite reacts fast with oxyhemoglobin (oxyHb; k2=2 x 10(4) M(-1) s(-1) at 37 degrees C and pH 7.4) and isomerizes to nitrate. Herein, we investigated whether, in spite of the fast diffusion and consumption of extracellularly added peroxynitrite by intraerythrocytic oxyHb, peroxynitrite-dependent radical processes could occur at the RBC membrane, focusing on tyrosine nitration. For this purpose, the hydrophobic tyrosine analogue N-t-BOC-L-tyrosine tert-butyl ester (BTBE) was successfully incorporated for the first time to a biological membrane, that is, RBC membrane, with incorporation yields approximately 1-3 x 10(7) molecules per RBC. The membrane integrity of BTBE-containing RBC was not significantly altered after BTBE incorporation as demonstrated by permeability studies. The probe was then used to study peroxynitrite-dependent reactions. The addition of peroxynitrite to BTBE-containing RBC suspensions resulted in BTBE nitration and dimerization to 3-nitro-BTBE and 3,3'-di-BTBE, respectively, indicative of peroxynitrite-derived radicals reactions in the membrane. Peroxynitrite addition to RBC also caused tyrosine nitration of membrane-associated proteins. The free radical nature of the process was also shown by the detection of protein-derived radicals by DMPO-immunospin trapping. While the presence of extracellular CO2 was potently inhibitory of intracellular oxyHb oxidation, membrane protein and BTBE nitration by peroxynitrite at <or=500 microM were, on the other hand, generally enhanced, supporting the participation of peroxynitrite-derived radicals (i.e., nitrogen dioxide and carbonate radicals) in the nitration process. Studies with erythrocyte ghosts having different contents of re-encapsulated oxyHb confirmed the role of intracellular oxyHb as a sink of nitrating species, as BTBE nitration and dimerization decreased with the increase in hemoglobin concentration. In summary, with the aid of a hydrophobic tyrosine analogue, our results show that peroxynitrite can mediate free radical-dependent nitration and dimerization processes in the RBC membrane, in spite of the significant peroxynitrite scavenging activity of oxyHb inside the RBC.