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

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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.

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Abbreviations

GndCl/β-ME:

a mixture of 6 M guanidine hydrochloride and 0.24 M β-mercaptoethanol

Hr:

hemerythrin

Hb:

hemoglobin

MW:

molecular weight

PBS:

phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 12 mM NaH2PO4, pH 7.4)

PEG:

polyethylene glycol

MM(PEG)12 :

sulfhydryl-reactive methyl-PEG-maleimide

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

TCEP:

Tris (2-carboxyethyl) phosphine hydrochloride

Tris/NaCl:

20 mM Tris-HCl, 150 mM NaCl, pH 7.4 buffer

TMS:

(Methyl-PEG12)3-PEG4-Nhydroxysuccinimide ester

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Acknowledgements

Drs. Francisco Carrascoza (BBU) and Donald M. Kurtz, Jr. (University of Texas at San Antonio, U.S.A.) are thanked for helpful discussions.

Funding

Funding from the Romanian Ministry of Education and Research (project PN-III-P4-ID-PCCF-2016-0142) is gratefully acknowledged.

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Authors and Affiliations

  1. Faculty of Chemistry and Chemical Engineering, Cluj-Napoca, Romania

    Mariann-Kinga Arkosi, Augustin C. Mot & Radu Silaghi-Dumitrescu

  2. Department of Biology 2, “Babes-Bolyai” University, 1 Mihail Kogălniceanu str, Cluj-Napoca, 400028, Romania

    Iulia Lupan & Miruna Georgiana Ghinia Tegla

Authors
  1. Mariann-Kinga Arkosi
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  2. Augustin C. Mot
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  4. Miruna Georgiana Ghinia Tegla
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  5. Radu Silaghi-Dumitrescu
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Contributions

MKA, ACM, IL, MGGT performed the experimental work. MKA and ACM prepared the Figures and Table. ACM and RSD wrote the main manuscript text. All authors reviewed the manuscript.

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Correspondence to Radu Silaghi-Dumitrescu.

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Arkosi, MK., Mot, A.C., Lupan, I. et al. Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes. Protein J 42, 374–382 (2023). https://doi.org/10.1007/s10930-023-10118-4

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