Fig. 16. EPR spectra from the solution of DNIC with neocuproine in 15 mM HEPES buffer, pH 7.0 (a) followed by pH increasing to 8.0 (b). Spectrum c was obtained by subtracting spectrum b from spectrum a. Recordings were made at 77 K .
inhibits the decomposition of RS—NO. This is usually interpreted as proof that Cu+ ions dominate this decomposition. In fact, the inhibition by neocuproine may be attributable to the sequestration of ferrous iron, which is also an efficient catalyst for the decomposition of RS—NO . The possibility of this alternative mechanism should be kept in mind when considering the pool of S-nitrosothiols in biological samples. More details of these reactions can be found in Chapters 12 and 13 of this book.
Two methods can be used for the synthesis of DNIC bound with proteins that do not contain intrinsic iron. The first is similar to the recipe for the formation of DNIC with low-molecular-weight DNIC (see above) and involves the treatment of the solution of proteins with gaseous NO in the presence of ferrous ions. The second method is the addition of low-molecular-weight DNIC to the solutions of proteins [54,72,73]. The protein-bound thiols also have high affinity for the Fe(NO)2 moiety of DNIC, and replace the small thiols on a large fraction of the dinitrosyl iron moieties. The exchange results in the formation of protein-bound DNIC. This second pathway is even easier for DNIC with non-thiol ligands: The weaker binding of such DNICs facilitates the transfer of all Fe(NO)2 moieties to protein. Fig. 17 demonstrate EPR spectra from DNIC-bovine serum albumin (DNIC-BSA) synthesized by these two methods.
Fig. 17. EPR spectra of bovine serum albumin (BSA) (1 mM) in 15 mM HEPES buffer, pH 7.4 at room temperature. (curve a) After the addition of 1 mM DNIC with phosphate, (curve b) after the addition of 1 mM FeSO4+ NO gas, (curve c) after adding 5 mM cysteine to preparation (a), (curve d) is spectrum (curve c) after subtraction of a small residual signal from Cys-DNIC, (curve e) after adding 1 mM ammonium sulfate to sample (a), (curve f) reference spectrum of 0.1 mM Cys-DNIC at 77 K and (curve g) reference spectrum of 0.1 mM Cys-DNIC at room temperature .
The shape of the spectra shows that the anisotropy of g-factor and HFS remain unchanged over the whole temperature range from 77 K to ambient [54,72,73]. This shows unambiguously that the DNICs are immobilized, in this case by being anchored to a large protein globule. The EPR signal from DNIC-BSA shows rhombic symmetry with g-factors 2.05, 2.03 and 2.014 [72,73].
This symmetry is lower than expected for DNIC with low-molecular-weight thiols, which have axial symmetry (see above). This low symmetry is plausibly rationalized by BSA molecule contributing only one thiol group to the DNIC. Second ligand L could be an intrinsic non-thiol amino acid residue like histidine. Thus, the different nature of the two anionic ligands could lower the symmetry of the complex from axial to rhombic. The idea is strongly supported by the observation fact that the symmetry may be upgraded to axial by the addition of cysteine to the DNIC-BSA solution (Fig. 17, curves c,d). This transformation was apparently due to the replacement of the protein-bound L ligand by cysteine in
DNIC (Scheme 2), the cysteine being free to assume its optimal orientation for binding to the iron .
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