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

• Published in: The protein journal Vol. 42; no. 4; pp. 374 - 382
• Main Authors: Arkosi, Mariann-Kinga, Mot, Augustin C., Lupan, Iulia, Tegla, Miruna Georgiana Ghinia, Silaghi-Dumitrescu, Radu
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-08-2023; Springer
• Subjects: Animal Anatomy; Biochemistry; Bioorganic Chemistry; Chemistry; Chemistry and Materials Science; Cysteine; Hemoglobin; Histology; Iron compounds; Morphology; Organic Chemistry; Polyethylene glycol; Spectrum analysis; Hemoglobin; PEGylation; Oxygen transport; Hemerythrin; Blood substitutes; Site-directed mutagenesis
• ISSN: 1572-3887; 1875-8355
• DOI: 10.1007/s10930-023-10118-4

Due to its ability to reversibly bind O
2
, 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.