Effect of covalent labeling of dextran-benzenehexacarboxylate on hemoglobin.

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.

Low oxygen affinity derivatives of human hemoglobin by fixation of polycarboxylic dextran to the oxyform.

Solutions of modified adult human hemoglobin (Hb) have potential applications as physiological oxygen carriers. The chemical modification that has been the most studied during the last few years is the cross-linking of the protein between its two alpha beta dimers, in order, first, to hamper their diffusion through the kidney and therefore increase the plasma persistence of Hb, and second, to decrease its oxygen affinity. However, despite the cross-linking, the vascular retention time is only increased by a factor of three, and a supplementary modification of cross-linked Hb is needed in order to further improve its in vivo half-life. The Hb derivatives described in this paper were obtained by the covalent fixation of benzene tetracarboxylate-substituted dextran onto oxyHb. The resulting conjugates all exhibited a higher P50 than native Hb. The experiments carried out in the presence of inositol hexaphosphate showed that the allosteric sites of Hb molecules were occupied by the polymeric reagent. The important decrease in the Bohr effect and the lack of the Cl- effect on the oxygen-binding properties proved that the Val 1 alpha residue was also substituted. Finally, the ability of some conjugates to unload as much O2 as blood, together with their other properties, make them quite promising candidates as red cell substitutes.

A potential blood substitute from carboxylic dextran and oxyhemoglobin. I. Preparation, purification and characterization.

Human adult hemoglobin (Hb) from peripheral venous blood was covalently linked, under its oxygenated form, to a dextran polymeric effector to yield conjugates with low oxygen affinity. The polymeric effector was synthesized by directly coupling benzene tetracarboxylic anhydride to dextran (MW approximately 10,000) under mild conditions. The reaction parameters were chosen so that the resulting polymeric derivative was only very little cross-linked and possessed benzene tricarboxylate site concentrations lesser than 1 site for 10 glucose units, as it was found that for higher concentrations the polymer exhibited anticlotting properties. The reaction between oxyHb and the polymer (dex-BTC) was carried out in water in the presence of a water-soluble carbodiimide (EDCI). By optimization of the reaction conditions, a dextran-Hb conjugate with a P50 of 21 torr (37 degrees C, 50mM Bis-tris buffer pH 7.4, 0.14M NaCl, 40 mM glucose) and a potential O2 release of 0.30 ml/g Hb between oxygen partial pressures of 100 and 40 torr, was obtained. The viscosity and oncotic pressure of a solution containing about 6% of conjugated Hb were respectively 1.8 cSt (37 degrees C) and 28 torr (25 degrees C). This solution could be stored frozen at -20 degrees C for a long time, without modification of its properties.

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.

Effect of dextran benzene polycarboxylates on the functional properties of human hemoglobin.

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.