The green nitrihemoglobin (α2β2 tetramer, NHb) was prepared by the aerobic reaction of excess nitrite with human hemoglobin A0 under mildly acidic conditions. A rate equation was determined and found to depend on nitrite, hydrogen ion and oxygen concentrations: –d[HbNO2]/dt = (k1 + k2(Ka[HNO2])[O2]1/2)•[ HbNO2], where k1 = 2.4 (±0.9) × 10-4 s-1, k2 = 1 (±0.2) × 105 M-5/2s-1 and Ka is the acid dissociation constant for nitrous acid (4.5 × 10-4 M). Also, the chemical properties of NHb are compared to those of the normal hemoglobin (including the addition products of both common oxidation states with exogenous ligands, the alkaline transitions of the ferric forms and the oxygen binding characteristics of the ferrous forms) and were found to be nearly indistinguishable. Therefore, the replacement of a single vinyl hydrogen with a nitro group on the periphery of each macrocycle in hemoglobin does not significantly perturb the interaction between the hemes and the heme pockets. Since non-photochemical reaction chemistry must necessarily be most dependent on electronic ground states, it follows that the clearly visible difference in color between hemoglobin A0 and NHb must be associated primarily with the respective electronic excited states. The possibility of NHb formation in vivo and its likely consequences are considered.

Abstract5-Fluoroanthranilic acid (FAA)-resistant mutants were selected in homothallic diploids of three Saccharomyces species, taking care to isolate mutants of independent origin. Mutations were assigned to complementation groups by interspecific complementation with S. cerevisiae tester strains. In all three species, trp3, trp4 and trp5 mutants were recovered. trp1 mutants were also recovered if the selection was imposed on a haploid strain. Thus, FAA selection may be more generally applicable than was previously described.