Daniel Sacco

Interaction of haemoglobin with dextran sulphates and the oxygen-binding properties of the covalent conjugates

Abstract The interactions between dextran sulphates of various molecular weights with both oxy- and deoxyhaemoglobin were studied by a potentiometric method and by measurement of their oxygen-binding parameters. These polyanionic polymers were shown to act as macromolecular effectors: in fact they bind to the liganded haemoglobin by means of a large number of relatively weak sites, whereas they strongly bind to the unliganded protein by means of a tight interaction inside the diphosphoglycerate binding cavity, which allowed the determination of the average binding stoichiometries of the dextran sulphate-deoxyhaemoglobin complexes. In both cases, the higher the molecular weight of dextran sulphate, the weaker the interaction, probably because of the steric hindrance, which hampers the approach of the protein by the polymer. In the light of these results, the conditions of the covalent fixation of dextran sulphate onto haemoglobin were defined, in order to obtain covalent conjugates with low oxygen affinity.

Interaction of a macromolecular polyanion, dextran sulfate, with human hemoglobin

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 β‐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.

A re-investigation of the phosphorylation of dextran with polyphosphoric acid: Evidence for the formation of different types of phosphate moieties

Abstract The products of phosphorylation of dextran with polyphosphoric acid have been re-investigated by gel filtration, potentiometric titration, and 31P-n.m.r. spectroscopy. Mainly (80–88%) alkyl phosphates are formed together with alkyl diphosphates and dialkyl phosphates, the percentages of which depended on the duration of phosphorylation. Mild acid treatment of the crude samples hydrolyzed the diphosphates and gave products with >95% of monophosphate structures.

Effect of polyanionic polymers on haemoglobin oxygen-binding properties: application to the synthesis of covalent conjugates with low oxygen affinity

Abstract Polyanionic water-soluble polymers containing sulphate, phosphate and polycarboxylate groups were synthesized. These compounds, when simply added to haemoglobin solutions, were shown to lower the affinity of the protein for oxygen. Their influence on oxygen affinity was regarded as the result of a specific interaction of the polymer anionic groups inside the 2,3-diphosphoglycerate-binding site of deoxyhaemoglobin. On the other hand, these polymers were linked to deoxyhaemoglobin to give covalent conjugates also exhibiting an oxygen affinity lower than that of free haemoglobin in the presence of 2,3-diphosphoglycerate, its natural effector, which means that after fixation, the polyanionic polymers are still acting as effectors.

Covalent fixation of hemoglobin to dextran phosphates decreases its oxygen affinity.

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.

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 bothαΒ 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 intoαΒ 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.

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.

Liquid chromatography of cephalosporin C and α-amino acid mixtures on polyfunctional polystyrene resins

Abstract The industrial separation of cephalosporin C from fermentation broths is usually achieved by liquid chromatography using ion-exchange resins, or adsorption on active carbon or neutral macroreticular sorbents. Unfortunately, the efficiency is low because of the low capacity or/and the poor selectivity of the resins used. Some new sorbents were synthesized by coupling three polyfunctional ligands (e- l -lysine, glycyl-e- l -lysine and diglycyl-e- l -lysine) on divinylbenzene-cross-linked polystyrene resins, and tested with an artificial aqueous mixture of cephalosporin C and α-amino acids. These stationary phases exhibit a selective affinity towards the antibiotic compared with the α-amino acids. In order to elucidate the mechanism of this adsorption, the influence of the ionic strength of the eluent on the retention of the different compounds used was investigated. This preliminary study allowed us to define optimal conditions for subsequent application to the preparative separation of cephalosporin C.