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.

Molecular Insight into the Protein–Polymer Interactions in N-Terminal PEGylated Bovine Serum Albumin

“…In 2011, Pai et al carried out small-angle neutron scattering (SANS) studies on mono-PEGylated lysozyme and mono-PEGylated human growth hormone and found that conjugated PEG with a molecular weight of 20 kDa acts as a random coil adjacent to the protein. However, a similar study performed in 2015 by Le Coeur et al showed that the polymer conformation in di-PEGylated hemoglobin depends on the molecular weight of the conjugated PEG chain . They demonstrated that the shroud-like conformation is favored when the PEG molecular weight is higher than 10 kDa.…”

Insight into Protein–Polymer Conjugate Relaxation Dynamics: The Importance of Polymer Grafting

“…The cell‐free Hb has long been proposed as a potential blood substitute but the clinical use remains hard due to problems related to inefficiency and toxicity, while, in contrast, PEGylated Hb greatly improved its performance. [ 21 ] Myoglobin is a small globular protein, with the same metal cofactor and basic tertiary arrangement of hemoglobin. The smaller dimensions of myoglobin, facilitate the synthetic preparation and purification of the samples.…”

Unraveling the Solution‐State Supramolecular Structures of Donor–Acceptor Polymers and their Influence on Solid‐State Morphology and Charge‐Transport Properties

“…This leads to the fact that each atom has specific neutron scattering cross‐section (Figure b). To increase the signal‐to‐noise ratio, hydrogenated polymer in a deuterated solvent system is selected to lower the incoherent scattering background because the scattering length for hydrogen and deuterium are greatly different ( b H = −3.739 × 10 −13 cm and b D = 6.671 × 10 −13 cm) . During the SANS measurement, we used deuterated 1,2‐dichlorobenzene‐ d 4 (ODCB‐ d 4 ) and toluene‐ d 8 as the solvents.…”