Demonstration of intermediates between deoxy and carbonmonoxy hemoglobins by anaerobic high-performance liquid chromatography

AbstractIntermediary ferrous hemoglobins (Hb) partially liganded with carbon monoxide (CO) were separated using an anaerobic high-performance liquid chromatography system equipped with a cation-exchange column at room temperature. Separations were achieved by applying various mixtures of oxy- and CO-Hb A to the column. The mobile phase contained 3 mM sodium dithionite which deoxygenated the oxy hemes but left the CO hemes intact, thereby producing hemoglobins bound with various numbers of CO molecules. Spectrophotometric analyses of the eluent showed that hemoglobins were eluted in the order of deoxy hemoglobin, Hb(CO), Hb(CO)2, Hb(CO)3 and Hb(CO)4