Chapter 1

Oxygen Binding Properties and Tetrameric Stability of Hemoglobins From the Snakes Crotalus Durissus Terrificus and Liophis Miliaris

Fábio Renato Lombardi, Maristella Conte Anazetti, Giovanni César Santos, Patrícia Peres Polizelli, Johnny Rizzieri Olivieri, Walter Filgueira de Azevedo and Gustavo Orlando Bonilla-Rodriguez

Abstract

Hemoglobins (Hbs) from the South American snake Liophis miliaris undergo a reversible oxygen-linked dissociation phenomenon α<sub>2</sub>β<sub>2</sub> + 4O<sub>2</sub> ↔ 2αβ(O<sub>2</sub>), due to substituted glutamate residues (43β and 101β) at the α<sub>1</sub>β<sub>2</sub>- interface, according to Matsuura et al., 1989. This work reports the oxygen-binding properties and analysis of tetrameric stability from the major hemoglobins of two snakes: a terrestrial species (Crotalus durissus terrificus), and a semiaquatic one, L. miliaris. The major hemoglobins from both species were analyzed by classic functional experimental approaches and other analytical techniques: dimer-tetramer association constant by analytical gel filtration chromatography and small angle x-ray scattering (SAXS). In addition, we performed molecular modeling of the hemoglobin from L. miliaris. The functional results display cooperative oxygen binding, alkaline Bohr Effect, and conformational transitions, a behavior which is compatible with tetrameric Hbs. Also, the estimated dimer-tetramer association constant <sup>4</sup>K<sub>2</sub>, radius of gyration, and maximum dimension exhibit similar compatibility. A molecular modeling of the structure of L. miliaris Hb, on the other hand, allows us to conclude that the residues Glu43β(CD2) and Glu101β(G3) are not essential in the maintenance of the tetrameric form, supporting our findings and allowing us to rule out the hypothesis proposed in the literature for the Hb of Liophis miliaris and other species of snakes. Therefore, the results reported in the paper suggest that the major Hbs of Liophis miliaris and Crotalus durissus terrificus are stable tetramers in vivo and in vitro.

Total Pages: 3-30 (28)

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