Fig. 18. EPR spectra from DNIC-bovine serum albumin (BSA) from initial preparation (curve a), after treatment with 1% dodecyl sulfate (curve b), and after its incorporation into reversed micelles formed from aerosol OT at the ratio between the amounts of water and aerosol ranging from 11 to 44 (curves c,d). Recordings were made at ambient temperature [73].

Non-heme DNIC is probably formed but remains unobservable because the EPR spectrum is dominated by the absorption from the ferrous nitrosyl-heme. But several chemical pathways exist which induce the formation of DNIC while avoiding nitrosylation of the heme: The addition of phosphate-DNIC ([phosphate] = 1 mM) to 1 mM Hb solution induces the formation of protein-bound DNIC, DNIC-Hb [73] (Fig. 19, curve a).

Fig. 19. EPR spectra from 1 mM solutions of horse hemoglobin after addition of 1 mM of DNIC with phosphate, including 56Fe (curve a) or 57Fe (curve b) or after addition of 1 mM DMIC with cysteine (curve c) or glutathione (curve d), including 56Fe. Recordings were made at ambient temperature [73].

The reaction proceeds presumably via transfer of the Fe-(NO)2 structure and does not involve the release of free NO. At room temperature, this complex shows an axially symmetric EPR spectrum, as distinct from the rhombic symmetry of DNIC-BSA. At 77 K, the EPR spectrum of DNIC-Hb coincides with that of Cys-DNIC in frozen solution. Incorporation of 57Fe into DNIC-Hb induces significant broadening of the EPR spectrum (Fig. 19, curve b). The DNIC-Hb complex did include thiol ligands, since treatment with mercurate resulted in the decomposition of the complex. Exposure of DNIC-Hb to dithionite resulted in the formation of paramagnetic ferrous heme-nitrosyl complexes. Such complexes have low spin (5 = 1/2) configuration {Fe(NO)}7 and are unambiguously identified by EPR signal with extreme components at g = 2.07 and 1.98 and triplet HFS at g = 2.01 described earlier elsewhere [74]. Such heme-nitrosyl complexes are also slowly formed if DNIC-Hb is kept under anaerobic conditions for a long time (longer than an hour). It shows that NO has a higher affinity for ferrous heme iron than for the non-heme iron in DNIC-Hb.

DNIC-Hb could also be obtained by incubating ferric hemoglobin with Cys-DNIC. In this process, Cys-DNIC was completely consumed [72]. However, the yield of protein-bound DNIC was considerably lower than that in the hemoglobin incubation with phosphate-DNIC (Fig. 19, curve c). DNIC-glutathione were found to be stable and failed to transfer the Fe-(NO)2 moiety into the hemoglobin. At room temperature, the solution retained the motionally narrowed EPR spectrum of DNIC-glutathione, and no signal from DNIC-Hb was observed [73] (Fig. 19, curve d).

DNIC-phosphate was also successfully applied to generate DNIC anchored on apo-metallothionein (apo-Mt) isolated from horse kidney [73]. The EPR signal from this complex coincided in shape and g-factor values with the EPR signal from frozen solution of DNIC with cysteine (Fig. 20, curve a). The shape of DNIC-apo-Mt signal remained unchanged

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