A simple protocol for the synthesis of dinitrosyl iron complexes with glutathione: EPR, optical, chromatographic and biological characterization of reaction products.

The diamagnetic binuclear form of dinitrosyl iron complexes (B-DNIC) with glutathione can be easily synthesized in the air at ambient temperature. The synthetic protocol includes consecutive addition to distilled water of glutathione, which decreases the pH of the test solution to 4.0, a bivalent iron salt (e.g., ferrous sulphate) and sodium nitrite at the molar ratio of 2:1:1, with a subsequent increase in pH to neutral values. Under these conditions, the amount of B-DNIC formed is limited by initial nitrite concentration. In the novel procedure, 20mM glutathione, 10mM ferrous sulfate and 10mM sodium nitrite give 2.5mM B-DNIC with glutathione, while 5mM glutathione remains in the solution. Bivalent iron (5mM) is precipitated in the form of hydroxide complexes, which can be removed from the solution by passage through a paper filter. After the increase in рН to 11 and addition of thiols at concentrations exceeding that of DNIC tenfold, B-DNIC are converted into a mononuclear EPR-active form of DNIC (M-DNIC) with glutathione. B-DNIC preparations synthesized by using new method contain negligible amount of nitrite or S-nitrosoglutathione as a contaminations. All the steps of DNIC synthesis were characterized by using optical, EPR and HPLC methods. A long-lasting hypotensive action of DNIC formed was demonstrated.