Measurements of HbNO in human RBCs by a modified EPR subtraction procedure.

<p>(A) Typical EPR spectra of free radicals in RBCs left for 45 min. in open air at room temperature: the individual model spectrum of free radicals in RBCs (<i>lowest spectrum, a–b</i>) was obtained after subtraction (subtraction I) of the EPR spectrum of RBCs treated with ascorbic acid (AA, 10 mmol/L, for the last 15 minutes) (b), from the EPR spectrum of RBC treated similarly with solvent (a). (B) Measurements of HbNO level in RBCs isolated from venous blood by EPR spectroscopy using subtraction (subtraction II) of individual free radical signal. The high field components of the EPR spectrum of RBCs frozen immediately after sampling (upper spectrum, a′) are shown at diapason 3250–3350 Gauss. The final EPR spectrum (a′–b′) shows the hf components of g_z HbNO signal after subtraction of the model EPR spectrum of free radicals (b′), divided by the correspondent proportional scale factor (gain = 6.23×10⁴); a typical EPR spectrum of RBCs, frozen after incubation with NO-donor (Dea-NONOate, 50 µmol/L) under low O₂ level (gain = 0.62×10⁴) is shown for comparison (d). The peak-to-peak amplitude of the 1st hf component (A(I)) of the triplet hf structure of the EPR spectrum, attributed to 5-coordinate α-HbNO, was used for quantitation.