Cloning and sequencing of hemoglobin II from the fish matrinxã, Brycon cephalus.
Poy, C.D.1, Babeto, E2, Souza, PC1, Souza, F.P.2, Leopoldino, A.M.3, Simone S.G.4 Rahal, P.2, Bonilla-Rodriguez, G.O.5
1 Pós-graduação em Biofísica Molecular, 2 Depto. de Biologia, IBILCE-UNESP, 3 FAMERP, São José do Rio Preto SP, 4 FIOCRUZ, Rio de Janeiro, 4 Depto. de Química e Ciências Ambientais, IBILCE, UNESP, São José do Rio Preto, SP, Brasil.
Oxygen transport in vertebrates is carried out by hemoglobin (Hb). It is a tetrameric assembly constituted by four globin chains, two alpha-like and two beta-like, having each one a prosthetic group responsible for O2 binding. Among fishes, O2-transport is usually performed by several iso-hemoglobins, occasionally with significant differences concerning their primary sequences and functional properties, influencing oxygenation and probably granting access to different environments. The objective of this work is to study the hemoglobins from this species in terms of their functional behavior and structure will allows us to understand the interplay between phosphate, proton and oxygen binding in the major components, Hb-I and II. For that purpose, after hemolysis we purified the hemoglobins by gel filtration and ion-exchange chromatography using DEAE-Sepharose. Purity of the isolated Hb was checked by isoelectric focusing. Partial sequences from Hb-II were obtained using Edman degradation, allowing to design primers for cDNA amplification. We obtained a fragment 438bp long, cloned and sequenced, showing to be similar to the alpha chain. Financial support: CNPq (CDP), FAPESP 03/00085-4 and CNPq (GOBR).
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