Apparent specific volume of human hemoglobin: Effect of ligand state and contribution of heme

The apparent specific volumes of human deoxy-, oxy-, met-, and CN-met hemoglobin (Hb) were measured with a vibrating tube densitometer. The values were calculated from the difference in density between protein solutions and solvents with which they were in dialysis equilibrium. The results obtained were very similar to the value for horse HbCO often used for sedimentation studies of Hbs. The apparent specific volumes of oxy- and CN-metHb are approximately 0.0020 cm3/g higher than those of deoxy- and metHb. This small reproducible difference could be due either to a small conformational difference between the liganded and unliganded species or to different interactions with components of the solvent. On the basis of these results, a simple method for the determination of the contribution of the heme to the apparent specific volume is proposed. The contribution can be estimated from the difference between the measured volume of each molecular species and that calculated from the amino acid composition.