Large-volume preparation of pyridoxylated hemoglobin with high P50

A technique is described for the preparation of a stroma-free pyridoxylated hemoglobin solution in volumes of up to 20 liters and hemoglobin concentrations of 18–20 g%. The methemoglobin concentration is 3–7%; the phospholipid content is 5–10% of lysate; and normal values for prothrombin time and activated partial thromboplastin time are obtained. Electrophoresis on cellulose acetate strips shows that 70% of the hemoglobin is covalently linked to pyridoxal-5′-phosphate. This proportion can be raised to greater than 85% (PLP fraction) by batch purification with DEAE-Sephadex. The final solution has an in vitroP₅₀ of 22–26 mm Hg (pH = 7.40, pCO₂ = 40 mm Hg, temperature = 37°C). Both the proportion of pyridoxylated to unmodified hemoglobin and the P₅₀ remain unchanged throughout the course of total exchange transfusion in the baboon.