Covalent Modifications of Hemoglobin by Nitrite Anion: Formation Kinetics and Properties of Nitrihemoglobin

Abstract

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.

Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules

“…The possibility that the nitrated heme originated by a substitution of a proton by NO 2 on a vinyl group was suggested by previous work on nitriMb (33) and nitriHb (34,35). These green Mb and Hb derivatives contain a 2-nitrovinyl group and are generated in vitro by exposing the proteins to excess NO 2 − .…”

Formation of nitri- and nitrosylhemoglobin in systems modeling the Maillard reaction

“…It is important that in the absence of methylglyoxal neither GSNO nor nitrite provoked metHb conversion to nitriHb. It was earlier shown that green nitriHb form appeared as result of metHb interacting with nitrite in aerobic conditions [30,31]. Otsuka et al [31] supposed that metHb was modified by nitrite complex with heme iron and nitration reagents such as N 2 O 3 and N 2 O 5 .…”

“…The amount of reduced Hb was determined through oxyHb (ε 576 = 14.6/mM/cm), formed in cuvette for spectrophotometric measurements in aerobic conditions. Formation of nitriHb was determined using an absorbance band of reduced hemochromogen (582 nm) in alkaline pyridine solution (30% pyridine solution in 0.1 M NaOH) [31].…”

“…In addition, in NO reactions with red-ox reactive products of Hb glycation RNS causing covalent modification of this protein can appear. Appearance of another Hb form -nitrihemoglobin (nitriHb) -was reported as a result of metHb interaction with nitrite in aerobic conditions [30,31]. In this reaction metHb turned to nitrimethemoglobin (nitrimetHb) because of covalent modification of porphyrin vinyl group as a result of electrophilic substitution reaction.…”

“…In this reaction metHb turned to nitrimethemoglobin (nitrimetHb) because of covalent modification of porphyrin vinyl group as a result of electrophilic substitution reaction. In some properties nitriHb was very similar to normal Hb (including the addition products of common oxidation states with exogenous ligands, the alkaline transitions of the ferric forms, and the oxygen binding characteristics of the ferrous forms) [30,31]. Although nitriHb is also a Hb form with N-containing ligand it has to be distinguished from nitrosylHb.…”