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The most frequent (90%) phenotype of the hemoglobin system of M. cephalus presented two major hemoglobins, the more anodal HbI accounting for approximately 70% of the total. The two hemoglobin components separated by ion-exchange chromatography were analyzed by reverse-phase HPLC and electrospray ionization-mass spectrometry which revealed a more complex pattern: HbI consists in four different globins, two b(namedb1andb3) and two co-eluting achains(a1 and a2); HbII consists in three globins, one bchain(namedb2) and the same a1 and a2 present in HbI. Theoxygen-binding properties of both hemoglobin components purified by DEAE cellulose were almost identical to those of the hemolysate: stripped hemoglobins howed a large Bohr effect which was enhanced by chloride ions and, a ta larger extent, by organic phosphates which, at acidic pH values gave rise to the Root effect. A series of oxygen-binding experiments at increasing GTP concentrations was carried out in order to compare GTP-binding activities in the absence and presence of physiological amount sofchloride

The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the alpha- and beta-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-alpha(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule.