Chemicals reacting with various forms of hemoglobin: biological significance, mechanisms, and determination

The clinical chemistry of various forms of hemoglobin occupies a large fraction of the total activities of hospital and forensic science laboratories. Some of the potential pitfalls are reviewed here along with an account of the accidental discovery of two novel chemical forms. Human or mouse red blood cells were exposed to excess sodium nitrite to convert the intracellular pigment to methemoglobin. When these were subsequently incubated in Krebs-Ringer-phosphate-glucose medium, pH 7.4 at 37 degrees C under nitrogen and in the presence of various concentrations of methylene blue, a blood pigment was generated in high yield which had unique properties. In lysates, the pigment was stable in air, and it could be maintained in liquid nitrogen for as long as a year without deterioration. The pigment had properties different from those of oxyhemoglobin, deoxyhemoglobin, methemoglobin, or carboxyhemoglobin. After separation by isoelectric focusing, the pigment gave a strong signal on electron paramagnetic resonance (EPR) spectroscopy. The other forms of hemoglobin given above are EPR-silent. The pigment was eventually identified as the nitrosylated valency hybrid species, (alpha 2+ beta 3+)2(NO)2. The corresponding species, (alpha 3+ beta 2+)2(NO)2, has similar properties. These species apparently owe their unusual stability in air to the presence of the oxidized subunits in the same tetramer.