The effects of the ionic strength and pH of the hemolyzing solution on the hemoglobin content of human erythrocyte ghosts were studied in phosphate buffers and found to have a pronounced influence upon hemoglobin binding in the ghosts. Buffer concentrations between 10 and 20 ideal milliosmolar (imOsm), at pH values 5.8 – 8.0, resulted in maximum hemoglobin removal from ghosts. The pH optimum for hemoglobin binding to ghosts was between 5.8 and 5.9 in a 20 imOsm buffer. The influence of these variables suggest an electrophysical interaction of hemoglobin with membrane constituents.

This study provides a basis for comparison of existing methods for ghost preparation, as well as a means for prediction of the conditions required for preparation of ghosts containing any desired amount of hemoglobin. Conditions were found that allowed the preparation of hemoglobin-free ghosts by single-stage hemolysis and washing. Hemoglobin-free ghosts were prepared in 20 imOsm phosphate buffer at pH 7.4. Essentially all the lipid was recovered in the ghosts, but non-hemoglobin nitrogen-containing substances were lost.

The pyridine hemochromogen method for hemoglobin determination was adapted for the measurement of very small quantities of hemoglobin through use of the Soret band (418 mμ) for absorbancy measurements.