Nitrite conversion to nitric oxide in red cells and its stabilization as a nitrosylated valency hybrid of hemoglobin.
The authors describe a method for the preparation, isolation and purification from human red blood cells of a stable form of hemoglobin containing both oxidized and reduced subunits. After isoelectric focusing across a pH gradient, it occupies the same position as the synthetically reconstituted or chemically generated species, (alpha 2+ beta 3+)2 (I). Unlike its synthetic or chemically generated counterpart, however, it (HbX) does not bind oxygen. A different method for the preparation in situ of the (alpha 3+ beta 2+)2 valency hybrid results in a pigment with chemical and spectral properties identical with those ascribed to its synthetically reconstituted counterpart, and both bind oxygen. HbX is generated in highest yield when red cells are incubated under N2 in the presence of glucose, methylene blue and nitrite for several hours. Its visible absorption spectrum differs from that reported for I, and it reacted very slowly with ferricyanide. Exposure to CO did not result in spectral shifts over that for HbX in air, but spectral shifts were produced with CO exposure of (alpha 3+ beta 2+)2. HbX had an inositol hexaphosphate difference-binding spectrum quite unlike that described for I. HbX also had a distinctive electron paramagnetic resonance spectrum that shifted after inositol hexaphosphate addition with a new three-line hyperfine pattern. Both spectra were characteristic for an unpaired electron in an iron-centered orbital with a hyperfine coupling to the nitrogen nuclear spin of a nitrosyl ligand on heme iron. The authors conclude that HbX is a form of I, but it has NO bound to its reduced subunits.
D004578:Electron Spin Resonance Spectroscopy; D005292:Ferricyanides; D006441:Hemoglobin A / Q000032:analysis / Q000235:genetics; D006454:Hemoglobins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing; D009569:Nitric Oxide / Q000097:blood*; D009573:Nitrites / Q000097:blood*; D010100:Oxygen / Q000097:blood; D055503:Protein Multimerization; D013053:Spectrophotometry
D005292:Ferricyanides; D006454:Hemoglobins; D009573:Nitrites; C008657:nitrosyl hemoglobin; D009569:Nitric Oxide; D006441:Hemoglobin A; D010100:Oxygen; C028033:potassium ferricyanide