NO-hemoglobin may be a light-sensitive source of nitric oxide both in solution and in red blood cells

Abstract

Hemoglobin in solution and inside red blood cells forms a complex with nitric oxide exhibiting a specific EPR signal both at room and liquid nitrogen temperatures. In the present paper it was shown that the nitrosyl complex of hemoglobin (NO-Hb) is photochemically sensitive and hence may serve as a source of free NO under He-Cd laser irradiation (441 nm). It was found that at laser light radiant power of 3.9 mW, room temperature and in the presence of oxygen, 50% decrease of NO-Hb EPR signal occurred at doses of 54, 30, and 18 kJ/m2 for NO-hemoglobin solution, hemolysed and intact erythrocytes, respectively. The detection of free NO produced as a result of NO-Hb photolysis was performed by means of a spin trap, nitronyl nitroxyl radical NNR, which in the presence of NO is transformed into imino nitroxyl radical (INR) showing different EPR signal. In isolated hemoglobin solution, 20 mM INR was accumulated under irradiation with the maximal dose of 700 kJ/m2. In intact cells the HbFe(2+)-NO photolysis and NO release occur with essentially higher efficacy. To produce 100 mM INR, a dose of 290 kJ/m2 was needed in erythrocyte lysates and 100 kJ/m2 in intact red blood cell suspension. Measurements of absorption spectra showed that in all systems studied (NO-Hb in solution, intact erythrocytes and hemolysed erythrocytes) NO-Hb concentration decreased after irradiation by 14-22% with simultaneous formation of methemoglobin. These observations show that NO-Hb may serve as a store of nitric oxide from which free NO can be released by intensive illumination.