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Open Access Research article

Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes

Daniela Coppola1, Stefano Bruno2*, Luca Ronda2, Cristiano Viappiani3, Stefania Abbruzzetti34, Guido di Prisco1, Cinzia Verde1 and Andrea Mozzarelli2

Author Affiliations

1 Institute of Protein Biochemistry, CNR, Naples, Italy

2 Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy

3 Department of Physics, University of Parma, NEST Istituto Nanoscienze-CNR, Parma, Italy

4 Department of Biotechnology, University of Verona, Verona, Italy

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BMC Biochemistry 2011, 12:66  doi:10.1186/1471-2091-12-66

Published: 20 December 2011

Abstract

Background

Conjugation of human and animal hemoglobins with polyethylene glycol has been widely explored as a means to develop blood substitutes, a novel pharmaceutical class to be used in surgery or emergency medicine. However, PEGylation of human hemoglobin led to products with significantly different oxygen binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity. These recent findings call for the biotechnological development of stable, low-affinity PEGylated hemoglobins with low NO dioxygenase reactivity.

Results

To investigate the effects of PEGylation on protein structure and function, we compared the PEGylation products of human hemoglobin and Trematomus bernacchii hemoglobin, a natural variant endowed with a remarkably low oxygen affinity and high tetramer stability. We show that extension arm facilitated PEGylation chemistry based on the reaction of T. bernacchii hemoglobin with 2-iminothiolane and maleimido-functionalyzed polyethylene glycol (MW 5000 Da) leads to a tetraPEGylated product, more homogeneous than the corresponding derivative of human hemoglobin. PEGylated T. bernacchii hemoglobin largely retains the low affinity of the unmodified tetramer, with a p50 50 times higher than PEGylated human hemoglobin. Moreover, it is still sensitive to protons and the allosteric effector ATP, indicating the retention of allosteric regulation. It is also 10-fold less reactive towards nitrogen monoxide than PEGylated human hemoglobin.

Conclusions

These results indicate that PEGylated hemoglobins, provided that a suitable starting hemoglobin variant is chosen, can cover a wide range of oxygen-binding properties, potentially meeting the functional requirements of blood substitutes in terms of oxygen affinity, tetramer stability and NO dioxygenase reactivity.