Preparation and physical characteristics of a hemoglobin solution modified by coupling to 2-nor-2-formylpyridoxal...

Stroma-free hemoglobin (Hb) solutions were prepared on a 20-I scale. In 3-I batches, the beta chains of hemoglobin were crosslinked with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP) that was synthesized on a gram scale. The coupling efficiency was 60 to 80 percent. The oxygen dissociation curve of these Hb/HbNFPLP mixtures was shifted to the right with P50 (PO2 for 50% saturation with oxygen) values of 26 to 38 torr versus values of 12 to 16 torr for the nonmodified hemoglobin solutions. Both the H+ Bohr factor and the Hill coefficient were lower for the Hb/HbNFPLP mixture than for the original hemoglobin solution. The oxygen-binding coefficient beta was the same for both types of Hb solutions. The viscosity and the colloid osmotic pressure of both solutions were also the same. During storage at 4 degrees C for 18 months, no precipitation or denaturation of hemoglobin was detectable in either solution. There was also no conversion of the modified hemoglobin molecules, HbNFPLP, to the native hemoglobin tetramers, dimers, or monomers. The percentage of methemoglobin remained at 5 percent for about 6 months; it increased to 26 percent over the next 12 months. The results indicate that intramolecular coupling of hemoglobin with NFPLP yields a stable product with physiologic oxygen-carrying properties.