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The covalent fixation of benzenehexacarboxylate (BHC) onto dextran was carried out according to several reaction schemes. The polyanionic polymers thus synthesized were capable of decreasing the oxygen affinity of hemoglobin by specifically interacting with the 2,3-diphosphoglycerate (2,3-DPG) binding site of the protein. The intensity of this effect was correlated to both the chemical structure of the polyanionic polymers and the BHC content in polymer. The polyanionic polymer, containing 0.035 mol BHC/g and presenting no cross-linking between its polymer chains, possessed the best effector properties. These properties were used to direct the covalent fixation of the dextran-benzenehexacarboxylate onto the phosphate binding site of the protein. The resulting hemoglobin was mainly substituted at the same time by one or more linked BHC onto both alpha beta dimers in the vicinity of the 2,3-DPG site. Thus, the modification of hemoglobin led to an increase in the hydrodynamic volume of each dimer sufficient to limit the diffusion of the conjugates through the kidney membrane, even if the conjugates had dissociated into alpha beta dimers. Compared to that of free hemoglobin, the oxygen affinity of the conjugates was significantly decreased. This type of covalent conjugate exhibited general properties quite suitable for use as blood substitutes.

The present study deals with the fixation of benzene polycarboxylates such as benzene hexacarboxylate (BHC) or benzene tetracarboxylate (BTC) onto dextran, with the aim of obtaining polyanionic polymers capable of decreasing the oxygen affinity of Hb by interacting with its phosphate binding site. The synthesis was carried out according to several reaction schemes. The polyanionic polymers presented the strongest effector properties, when the polyanionic molecule (BHC or BTC) were directly fixed onto the hydroxyl functions of dextran in the presence of a carbodiimide or by means of the benzene tetracarboxylic anhydride. The introduction of a spacer arm between the polymer and the polyanionic molecule led to the formation of crosslinking between chains of dextran. Among the synthesized polymers those which possess effector properties similar to those of 2,3-diphosphoglycerate (2,3-DPG) were bound onto oxyHb. The resulting covalent conjugates exhibited a low oxygen affinity and a molecular size quite fitted to a potential use as blood substitutes.

The interactions between various dextran phosphates and Hb (hemoglobin) were studied by measuring the oxygen-binding parameters of the mixtures. The effector properties of polymers were found to depend on the concentration of monoalkylmonophosphate groups on the polymers and also on their molecular weights. The covalent fixation of dextran phosphates bearing aldehydic groups to oxyHb and deoxyHb was carried out. The oxygen-binding properties of the conjugates thus obtained depended upon the initial form of the protein. Thus, only the conjugates synthesized from deoxyHb exhibited a low oxygen affinity, which means that, in this case, the linkages between the dextran phosphate and the protein allow a permanent interaction of the phosphate groups with amines of the 2,3-diphosphoglycerate binding site. The Hill coefficient values of these conjugates were smaller than that of free Hb, corresponding to a loss of the cooperativity of the protein upon fixation of polymers. However, as these new conjugates are capable of unloading more O2 than blood when subjected to oxygen pressures corresponding to physiological conditions, they can be regarded as potential erythrocyte substitutes.

Interactions of dextran sulfate with amino groups of oxy- and deoxyhemoglobin were followed by both potentiometric measurements between pH 6 and 7.3 and oxygen-binding studies. The uptake of protons observed upon addition of dextran sulfate to hemoglobin shows that the interaction with the deoxy form is strong and that the main site is probably located in the phosphate-binding beta-cavity, whereas the interaction with the oxy form is more diffuse, probably with a great number of relatively weak binding sites. The influence of dextran sulfate on the oxygen dissociation curve of hemoglobin confirms these findings, as the effect of the polymer is to lower hemoglobin affinity for oxygen to a great extent, which proves that it stabilizes the deoxy form more strongly than the oxy one.