title: Conformation of the Poly(ethylene Glycol) Chains in DiPEGylated Hemoglobin Specifically Probed by SANS: Correlation with PEG Length and in Vivo Efficiency.
abstract: Cell-free hemoglobin (Hb)-based oxygen carriers have long been proposed as blood substitutes but their clinical use remains tricky due to problems of inefficiency and/or toxicity. Conjugation of Hb with the biocompatible polymer poly(ethylene glycol) (PEG) greatly improved their performance. However, physiological data suggested a polymer molecular weight (Mw) threshold of about 10 kDa, beyond which the grafting of two PEG chains no longer improves efficiency and nontoxicity of diPEG/Hb conjugates. We used small-angle neutron scattering and contrast variation, which are the only techniques able to probe separately the conformation of PEG chains and Hb protein within the complex, to investigate the role of PEG chain conformation in diPEGylated Hb conjugates as a function of the polymer Mw. We found out that the structure of Hb tetramer is not modified by the polymer grafting. Similarly, with a constant grafting of two chains per protein, there is no significant change of the Gaussian conformation between free and grafted PEG below ∼10 kDa, the complex being well described by the "dumbbell" model. However, beyond that threshold, the radius of gyration of grafted PEG is significantly smaller than that of the free polymer, showing a compaction of the PEG chains, either in the "dumbbell" model or in the "shroud" one. In the latter model, the polymer may be wrapped on the surface of the protein spreading a protective "shielding" effect over a larger fraction of the protein. Both proposed models are in good agreement with the physiological data reported in the literature.
authors.affiliation: †Laboratoire Léon-Brillouin (LLB), UMR 12 CEA-CNRS, CEA-Saclay, F-91191 Gif-sur-Yvette CEDEX, France.
authors.forename: Clémence
authors.initials: C
authors.lastname: Le Cœur
authors.affiliation: †Laboratoire Léon-Brillouin (LLB), UMR 12 CEA-CNRS, CEA-Saclay, F-91191 Gif-sur-Yvette CEDEX, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Combet
authors.affiliation: †Laboratoire Léon-Brillouin (LLB), UMR 12 CEA-CNRS, CEA-Saclay, F-91191 Gif-sur-Yvette CEDEX, France.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Carrot
authors.affiliation: ‡Forschungszentrum Jülich GmbH, Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Lichtenbergstrasse 1, D-85747 Garching, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Busch
authors.affiliation: †Laboratoire Léon-Brillouin (LLB), UMR 12 CEA-CNRS, CEA-Saclay, F-91191 Gif-sur-Yvette CEDEX, France.
authors.forename: José
authors.initials: J
authors.lastname: Teixeira
authors.affiliation: †Laboratoire Léon-Brillouin (LLB), UMR 12 CEA-CNRS, CEA-Saclay, F-91191 Gif-sur-Yvette CEDEX, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Longeville
mesh_terms: D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D033363:Neutron Diffraction; D011092:Polyethylene Glycols / Q000737:chemistry*; D053838:Scattering, Small Angle
chemical_list: D006454:Hemoglobins; D011092:Polyethylene Glycols