title: Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Coppola
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruno
authors.forename: Luca
authors.initials: L
authors.lastname: Ronda
authors.forename: Cristiano
authors.initials: C
authors.lastname: Viappiani
authors.forename: Stefania
authors.initials: S
authors.lastname: Abbruzzetti
authors.forename: Guido
authors.initials: G
authors.lastname: di Prisco
authors.forename: Cinzia
authors.initials: C
authors.lastname: Verde
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001802:Blood Substitutes / Q000737:chemistry / Q000378:metabolism*; D029941:Fish Proteins / Q000737:chemistry / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D010473:Perciformes / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001802:Blood Substitutes; D029941:Fish Proteins; D006454:Hemoglobins; D011092:Polyethylene Glycols; D010100:Oxygen