Characteristics of stroma-free hemoglobin prepared by crystallization

Stroma-free hemoglobin was prepared from outdated human red cells by crystallization. After hemolysis with water and toluene and low speed centrifugation, the solution was dialyzed against 2.8M phosphate buffer. Hemoglobin crystals formed within the dialysis casing and were washed with phosphate buffer. After being dissolved in water, dialyzed against kidney dialysis fluid, and sterilized by Millipore filtration, the hemoglobin solution obtained had normal serum potassium, sodium, and osmolality. Spectral maxima and minima were characteristic for oxyhemoglobin, and cellulose acetate electrophoresis showed a sharp, well-defined hemoglobin band and slight contamination with carbonic anhydrase. The crystallized hemoglobin solution showed no coagulant activity, and preparation from group A cells showed no blood group A activity by hemagglutination inhibition. Methemoglobin was less than 0.28 gm./dl. and did not increase with storage at refrigerated temperature for a period up to 6 months. The P50 ranged between 15 and 18 mm. Hg at pH 7.4 and n values were normal.