Selective polyethylene glycol attachment to hemerythrin for potential use in blood substitutes

Abstract

Abstract The oxygen carrying protein hemerythrin represents an alternative raw material to hemoglobin-based blood substitutes due to its reversible oxygen binding properties, relatively low redox reactivity and limited cytotoxicity. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, containing a single cysteine residue on the surface of each subunit of the hemerythrin octamer were engineered for specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was employed 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 was reasonably stable, displayed an oxygen affinity similar to that of the wild-type, and showed an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.