HEMOGLOBIN LINKED TO POLYANIONIC POLYMERS AS POTENTIAL RED-BLOOD-CELL SUBSTITUTES

A variety of chemical modifications of hemoglobin (Hb) have been proposed in order to transform it into cell-free oxygen carriers. These modifications are intended to increase the plasmatic half-life of the protein and to lower its affinity for oxygen. We have designed and prepared derivatives of dextran and polyoxyethylene functionalized so that, after their covalent fixation onto Hb, they can act as permanent effectors and thus lower the oxygen affinity of the protein while decreasing its renal excretion. The main characteristic of these functionalized polymers is that they possess a relatively high density of anionic groups. Benzene hexacarboxylate (BHC) and tetracarboxylate (BTC) linked to water-soluble polymers such as polyoxyethylene or dextran, yielded polymeric derivatives which, even after reaction with oxyHb, gave rise to conjugates with a lower oxygen affinity than the native protein. We showed on a conjugate obtained by the fixation of a BHC-monosubstituted polyoxyethylene onto oxyHb, that the low oxygen affinity was due to the preferential binding of the polymer-linked BHC to the beta-terminal valine residues. In the reaction of dextran-linked benzene tetracarboxylate (dex-BTC) with oxyHb, a lot of parameters had to be optimized in order to obtain conjugates well fitted to the purpose of blood substitute. Thus the polymer content in BTC and the amounts of reagents used for the reaction determined the oxygen-binding properties, the molecular weights and the biochemical characteristics of the conjugates, as well as the viscosity and oncotic pressure of the solutions. This optimization resulted in products which are now studied in-vivo.