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1.  Crystallization, preliminary X-ray diffraction studies and Raman microscopy of the major haemoglobin from the sub-Antarctic fish Eleginops maclovinus in the carbomonoxy form 
The major haemoglobin of the sub-Antarctic fish E. maclovinus is the first sub-Antarctic fish haemoglobin to be crystallized and its structural characterization will shed light on the molecular mechanisms of cold adaptation and the role of the Root effect in fish.
The blood of the sub-Antarctic fish Eleginops maclovinus (Em) contains three haemoglobins. The major haemoglobin (Hb1Em) displays the Root effect, a drastic decrease in the oxygen affinity and a loss of cooperativity at acidic pH. The carbomonoxy form of HbEm1 has been crystallized in two different crystal forms, orthorhombic (Ortho) and hexagonal (Hexa), and high-resolution diffraction data have been collected for both forms (1.45 and 1.49 Å resolution, respectively). The high-frequency resonance Raman spectra collected from the two crystal forms using excitation at 514 nm were almost indistinguishable. Hb1Em is the first sub-Antarctic fish Hb to be crystallized and its structural characterization will shed light on the molecular mechanisms of cold adaptation and the role of the Root effect in fish haemoglobins.
PMCID: PMC3001669  PMID: 21045316
cold adaptation; Eleginops maclovinus; haemichrome; haemoglobin; oxygen affinity; Raman microspectroscopy
2.  Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes 
BMC Biochemistry  2011;12:66.
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.
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.
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.
PMCID: PMC3268738  PMID: 22185675

Results 1-2 (2)