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. 2023 Aug;42(4):374-382.
doi: 10.1007/s10930-023-10118-4. Epub 2023 Apr 29.

Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes

Mariann-Kinga Arkosi  1 , Augustin C Mot  1 , Iulia Lupan  2 , Miruna Georgiana Ghinia Tegla  2 , Radu Silaghi-Dumitrescu  3
Affiliations

Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes

Mariann-Kinga Arkosi et al. Protein J. 2023 Aug.

Abstract

Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

Keywords: Blood substitutes; Hemerythrin; Hemoglobin; Oxygen transport; PEGylation; Site-directed mutagenesis.

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