Protective Effect of Dinitrosyl Iron Complexes Bound with Hemoglobin on Oxidative Modification by Peroxynitrite

Figure 1. Schematic image of dinitrosyl iron complex (DNIC) bound with tetrameric erythrocytic hemoglobin (Hb-DNIC).

Figure 2. Destruction of metHb-bound dinitrosyl iron complexes (Hb-DNICs) with peroxynitrite (ONOO−). The reaction mixture contained 0.2 M Na, K-phosphate buffer (pH 7.2) and 0.4 mM Hb-DNICs (Hb concentration). (A)—typical electron paramagnetic resonance (EPR) spectra recorded 4.5 min after adding various peroxynitrite concentrations to the reaction mixture: 1—Hb-DNICs without peroxynitrite, 2—Hb-DNICs + 1.25 mM (1:3.2), 3—Hb-DNICs + 2.5 mM ONOO− (1:6.25), 4—Hb-DNICs + 4.8 mM ONOO− (1:12), 5—Hb-DNICs + 5.8 mM ONOO− (1:14.5), the molar ratio of Hb or Hb-DNICs to ONOO− is shown in parenthesis. (B)—kinetics of Hb-DNICs degradation with the action of various ONOO− concentrations (results of five experiments).

Figure 3. Formation of metHb carbonyl derivatives under the impact of various concentrations of peroxynitrite. Reaction mixture contained—0.2 M Na,K-phosphate buffer (pH 6.8) and 0.15 mM Hb-DNICs (Hb concentration). Curves 1 and 3metHb, curves 2 and 4—Hb-DNICs, curves 3 and 4—with DTPA. The modification was carried out at the following concentrations of ONOO– in the reaction medium: 0 mM, 0.38 mM (1:2.5), 0.6 mM (1:4), 1 mM (1:6.5), 1.6 mM (1:10.6), 2.55 mM (1:17), 4.2 mM (1:28). The molar ratio of metHb or Hb-DNICs to ONOO- is shown in parentheses.

Figure 4. (A)—destruction of the heme group of methemoglobin under the impact of peroxynitrite. Curves 1 and 3—metHb, curves 2 and 4—Hb-DNICs, curves 3 and 4—with diethylenetriaminepentaacetic acid (DTPA). The composition of the reaction medium, including peroxynitrite concentration, is the same as in the legend to Figure 3. (B)—Changes in the state of the heme group in Hb-DNICs during the oxidation by peroxynitrite. Left—metHb, right—Hb-DNICs.

Figure 5. Pathways of nitration of tyrosine (A) and tryptophan residues (B). Scheme A also shows the formation of 3,3′-dityrosine dimer and 3-nitrosotyrosine.

Figure 6. Changes in fluorescence during the formation of dityrosines (A), and during the oxidation of tryptophan residues (B) under peroxynitrite action. Curve 1—metHb, 2—Hb-DNICs. The composition of the reaction medium including peroxynitrite concentration is the same as in the legend to Figure 3.

Figure 7. Influence of ONOO− on the amount of SH- groups of metHb detected by 10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-H-naphtho[2,1-b]pyran-2-carboxylic acid–methyl ester (ThioGlo1), which forms an adduct with maximum fluorescence at 500 nm at an excitation wavelength of 379 nm. The ratio of metHb to ThioGlo1 in the reaction medium is 1:8 mM. Curve 1—metHb, 2—Hb-DNICs. The composition of the reaction medium, including ONOO− concentration, is the same as in the legend to Figure 3.

Figure 8. Total amount of covalent aggregates of metHb subunits. According to the data of SDS electrophoresis in 12 % PAAG with dithiothreitol (DTT). * Differences were considered significant at p < 0.05.