ISOLATION OF INTERMEDIATE VALENCE HYBRIDS BETWEEN FERROUS AND MET HEMO GLOBIN AT SUBZERO TEMPERATURES

Quenching a [human] Hb solution partially saturated with CO into a hydro-organic solvent containing ferricyanide will produce under suitable conditions a population of partially oxidized and CO-bound Hb molecules. Since each Fe3+ heme carries 1 extra charge, it should be possible, in theory, to resolve the spectrum of intermediate compounds between Hb and CO, which was originally present in solution. The following are discussed: the development of a simple and rapid method to quench aqueous Hb solutions into a hydro-organic solvent at subzero temperatures; the determination of suitable experimental conditions to isolate valence hybrids between HbCO and methemoglobin by isoelectric focusing at temperatures as low as -25.degree. C; and the identification and isolation of all valence hybrids of different charge between HbCO and methemoglobin.