Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit.

Autor:	Cooper CE; School of Life Sciences, University of Essex, Colchester, United Kingdom., Bird M; Abzena Ltd, Cambridge, United Kingdom., Sheng X; Abzena Ltd, Cambridge, United Kingdom., Choi JW; Abzena Ltd, Cambridge, United Kingdom., Silkstone GGA; School of Life Sciences, University of Essex, Colchester, United Kingdom., Simons M; School of Life Sciences, University of Essex, Colchester, United Kingdom., Syrett N; School of Life Sciences, University of Essex, Colchester, United Kingdom., Piano R; Department of Medicine and Surgery, University of Parma, Parma, Italy., Ronda L; Department of Medicine and Surgery, University of Parma, Parma, Italy.; Institute of Biophysics, National Research Council, Pisa, Italy., Bettati S; Department of Medicine and Surgery, University of Parma, Parma, Italy.; Institute of Biophysics, National Research Council, Pisa, Italy., Paredi G; SITEIA.Parma, University of Parma, Parma, Italy., Mozzarelli A; Institute of Biophysics, National Research Council, Pisa, Italy.; Department of Food and Drug, University of Parma, Parma, Italy., Reeder BJ; School of Life Sciences, University of Essex, Colchester, United Kingdom.
Jazyk:	angličtina
Zdroj:	Frontiers in chemistry [Front Chem] 2021 Jul 26; Vol. 9, pp. 707797. Date of Electronic Publication: 2021 Jul 26 (Print Publication: 2021).
DOI:	10.3389/fchem.2021.707797
Abstrakt:	In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo . We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate. Competing Interests: CC and BR have patents granted and pending relating to modification of hemoglobin amino acids designed to enhance the performance of an oxygen therapeutic and are shareholders in a related company (CymBlood). Authors MB, XS, and JC were employed by the company Abzena Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2021 Cooper, Bird, Sheng, Choi, Silkstone, Simons, Syrett, Piano, Ronda, Bettati, Paredi, Mozzarelli and Reeder.)
Databáze:	MEDLINE
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