Resonance Raman in Vitro Detection and Differentiation of the Nitrite-Induced Hemoglobin Adducts in Functional Human Red Blood Cells.

This work presents in vitro studies of the functional, isolated human red blood cells (RBCs) treated with various concentrations of Na14NO2 and Na15NO2 with the use of resonance Raman spectroscopy (RRS) at two different laser excitations supported by absorption spectrophotometry (UV-vis). The products of the reaction between oxyhemoglobin (oxyHb) in isolated RBCs with NaNO2 were analyzed and identified in situ. The metHb-H2O was found to be the major product of this reaction; however, additional adducts were also clearly observed. Vibrational analysis allowed identification of various Hb3+NO2 species (Fe3+-O-N=O with O-binding mode of nitrite ion to the Fe3+ core and nitrovinyl adducts with 2-vinyl nitration favored over 4-vinyl nitration) as well as the Fe3+-NO adduct. In addition, we were able to visualize in situ the Hb-NO2 species inside functional RBCs with the use of Raman imaging.