Figure 1.

Production of iron nitrosylHb by addition of NO to variously oxygenated Hb. (A) EPR spectra of iron-nitrosyl Hb derivatives formed by incubation of 19 μM NO with 393 μM Hb at various degrees of oxygen saturation in 10 mM phosphate buffer, pH 7.4. The oxygen saturations for the largest through smallest EPR signals are 5.5, 32, 50, and 69%, respectively. Spectra show predominantly six coordinate α and β nitrosyl hemes, as typically observed for Hb in R state. (B) EPR spectra of iron-nitrosyl Hb derivatives formed by incubation of 55 μM NO with 380 μM Hb at various degrees of oxygen saturation in 100 mM phosphate, pH 7.4. The oxygen saturations for the largest through smallest EPR signals are 1, 15, 41, 60, and 80%, respectively. Spectra show a significant component of five coordinate α nitrosyl hemes (triplet structure) associated with Hb in T state. (C) Trials conducted with Hb in 10 mM phosphate, pH 7.4. The symbols are experimental results and the solid lines represent a best fit to the functional form for cooperative NO binding. Open diamonds, 393 μM Hb incubated with 19 μM NO; open circles, 350 μM Hb incubated with 15 μM NO plus 0.05% borate (added to bring the buffer concentration to 100 mM as in D); open squares, 365 μM Hb incubated with 15 μM NO and 1,190 units/ml SOD. (D) Trials conducted with Hb in 100 mM phosphate, pH 7.4. The symbols are experimental results, and the lines represent a best fit to the functional form for simple competition between oxidation and NO addition reactions (Eq. 4). Filled circles, 380 μM Hb incubated with 55 μM NO; filled squares, 375 μM Hb incubated with 7 μM NO. Application of the simple competition function to data of C or the cooperativity function to data of D gives an order of magnitude increase in χ².