G 242 225 207 203 194 191 1 1 t till

Fig. 26. EPR spectra of livers of mice maintained on a diet with nitrite [121] or nitrate (curve b) [12]. (curve c) Liver from control mice [121]. Recordings at 77 K. The EPR signals with g = 2.42, 2.25 and 1.91, g = 2.07, 2.03 or 1.94 are due to cytochrome P450, Hb-NO complexes, protein-bound DNICs or reduced iron-sulfur centers, respectively.

the signal to dominate the EPR spectra (Fig. 26). The frozen tissues showed additional EPR absorption from Hb-NO and iron-sulfur centers in the respiratory chain (cf Chapters 5 and 15 for more detail, respectively).

The yield of DNIC could be influenced by the administration of iron complexes to the nitrite-rich diet. The yields in liver increased by a factor 3-4 to ca 30 ^M/kg after adding iron citrate (0.02%) to the nitrited drinking water [121-126]. Such high yields were observed only upon feeding with the combination of nitrite and iron. If the iron supplement was removed from the nitrite-containing drinking water after 20 days, the intensity of the 2.03 signal in the liver of these animals began to fall slowly on a timescale of a week. After 10 days, the amount of the DNICs had fallen to 15 ^M/kg of wet tissue. The complexes g = 2.03

Fig. 27. Change of the shape of 2.03 signal from yeast cells (curve a) induced by treatment of yeast preparations with phenahtroline (curve b), ethylxanthogenate (curve c) or HgCl2 (curve d). Recordings at 77 K [9].

a b c d were also observed in the kidney, small intestines and blood although the yield was generally 5-10 times less than in the liver. In the heart, lungs or spleen, the DNICs remained below the detection limit of ca 0.1 ^M/kg due to masking of the 2.03 signal by other EPR signals.

The isotopic substitution of 56Fe (I = 0) by 57Fe (I = 1 /2) in the drinking water caused line broadening in the DNIC spectra of tissues [122,127]. The experiments proved that a significant fraction of DNIC contained iron from the dietary intake. After prolonged dietary intake, the signal was compatible with complete replacement of 56Fe by 57Fe in the complexes (Fig. 28). The yields in liver tissue reached the same maximal amount of 30 ^M/kg of wet liver tissue [122] observed before with unlabeled iron. Interestingly, subsequent exposure of the liver tissue to gaseous NO further increased the DNIC yield threefold [122]. At the same time, the linewidth decreased significantly and showed unequivocally that endogenous stores of 56Fe iron contribute to the formation of the DNIC complexes (Fig. 28).

The predominant incorporation of exogenous iron (57Fe) into DNICs allows to suggest that the Fe-NO groups forming part of the complexes appear in the blood or intercellular fluid. After being incorporated into the organ tissues, these groups link up with the thiol-containing proteins which also leads to the formation of 2.03 complexes [121-124]. We were able to prove feasibility of this formation by intraperitoneal injections of low-molecular DNIC-thiol. Such injections indeed significantly increased the yield of DNIC in all tissues [124-127].

Fig. 29 shows the evolution in time of 2.03 signal in frozen liver, kidney and blood from rats which were injected intraperitoneally with DNIC-cysteine [125]. The lineshape of the 2.03 signal from liver, kidney or blood as recorded 6 h after the injection of Cys-DNIC with 56Fe or 57Fe resembled that of the DNICs from the same organs extracted from rats maintained on drinking water with nitrite and iron (Fig. 30) [125]. This coincidence was also found in different species like rats, cats, guinea pigs, rabbits, mice and hamsters [125]. The lineshape remained unchanged upon thawing the samples to room temperature and proved that the DNICs were bound to protein.

Fig. 28. 2.03 signals in livers of mice maintained on drinking water with nitrite + 57Fe or 56Fe during 6 days (spectra a or b, respectively) followed with gaseous NO treatment (spectra c or d, respectively). Spectra (e,f) show 2.03 signals in liver homogenate preparations from control mice (without nitrate) added respectively with 57Fe or 56Fe followed with gaseous NO treatment. Recordings were made at ambient temperature. On right, amplifications in relative units. S is the width of the signal component at half amplitude (5 = 2.7 or 17 mT for DNIC-cysteine EPR signal with 57Fe or 56Fe, respectively) [122,124].

Fig. 28. 2.03 signals in livers of mice maintained on drinking water with nitrite + 57Fe or 56Fe during 6 days (spectra a or b, respectively) followed with gaseous NO treatment (spectra c or d, respectively). Spectra (e,f) show 2.03 signals in liver homogenate preparations from control mice (without nitrate) added respectively with 57Fe or 56Fe followed with gaseous NO treatment. Recordings were made at ambient temperature. On right, amplifications in relative units. S is the width of the signal component at half amplitude (5 = 2.7 or 17 mT for DNIC-cysteine EPR signal with 57Fe or 56Fe, respectively) [122,124].

Fig. 29. Left panel: Time variation of 2.03 signals in liver (curve a), kidney (curve b) and blood (curve c) of rats injected intraperitoneally with Cys-DNIC. Recordings were conducted at 1 (---), 3 (------) or 6 (-) h after injection. Right panel: 2.03 signals from Cys-DNIC in frozen solution (curve a), from liver (curve b); from kidney (curve c) and from blood (curve d). The tissues were taken 6 h after the injection of Cys-DNIC. The dashed line corresponds to Cys-DNIC with 57Fe, solid line to Cys-DNIC with 56Fe. Spectra were recorded at 77 K. (From Ref. [125].)

Fig. 29. Left panel: Time variation of 2.03 signals in liver (curve a), kidney (curve b) and blood (curve c) of rats injected intraperitoneally with Cys-DNIC. Recordings were conducted at 1 (---), 3 (------) or 6 (-) h after injection. Right panel: 2.03 signals from Cys-DNIC in frozen solution (curve a), from liver (curve b); from kidney (curve c) and from blood (curve d). The tissues were taken 6 h after the injection of Cys-DNIC. The dashed line corresponds to Cys-DNIC with 57Fe, solid line to Cys-DNIC with 56Fe. Spectra were recorded at 77 K. (From Ref. [125].)

g = 2.037 2.03 2.012 2.005 it it g = 2.037 2.03 2.012 2.005 it it

Fig. 30. 2.03 signals from liver (curve a), kidney (curve b) and blood (curve c) of rats, maintained on drinking water with nitrite and iron salts during 7 days. Recordings at 77 K [125].

At higher microwave frequencies, the anisotropy of the Zeeman interaction is better resolved. At Q-band frequencies (~30 GHz), the lineshape of the g = 2.03 signal in mouse liver showed a g-factor with rhombic symmetry (Fig. 31) [128].

This rhombicity is incompatible with the axial symmetry of DNIC with cysteine. Heating of the preparation to 60°C for 5 min changed the symmetry from rhombic to axial as found in DNIC with cysteine in frozen solution. The same transformation took place in samples stored at ambient temperature for 3-4 h. At X-band frequencies, the rhombicity of the Zeeman interaction is hardly noticeable. It only appears in the form of a small distortion near the central part of the 2.03 signal. Similar distortions appear in liver tissues from cat, guinea pig, rat or mouse (Fig. 32) and are attributed to rhombic distortion of the g-tensor

Figs. 29 and 30 show that blood samples contain DNIC with a slightly different EPR lineshape: In blood, the component at g| |has lower intensity than the common 2.03 signal

[125.126]. This form is characteristic of DNIC moieties anchored to Hb that was shown before for isolated DNIC-Hb (Fig. 19). This fact is in line with the proposition about the capability of low-molecular DNICs, at least their Fe(NO)2 groups penetrate through cell membranes followed by binding with thiol group of Hb or other proteins. The separation of rat blood indicated that the majority of the DNICs in blood was located in the RBC fraction rather than the plasma [125,126].

As mentioned before, Cys-DNIC shows axial symmetry in frozen aqueous solutions. Spectral distortions are observed in polar organic solvents like DMF, DMSO, tetramethylurea or

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