Caratterizzazione strutturale e funzionale dei sistemi emoglobinici di due specie di pesci (Mugil cephalus e Ophisurus serpens) e di due varianti emoglobiniche umane (HbRoma e HbF-SS-Monserrato)

The hemoglobin is a very important protein that in vertebrates binds reversibly the oxygen and transport it to the tissue through the circulatory sistem. The aim of this study is to contribute to the knowledge of structural-functional relationship of the hemoglobin molecule through structural and funcional characterisation of human hemoglobins natural variants and those present in different animal species. We report the identification and characterisation of two new hemoglobin variant, never described before, that we have named HbF-Monserrato-Sassari [Gγ93(F9) Cys Arg] and Hb Roma [β115(G17)Ala Val], and the hemoblobin systems of two species of fish, Mugil cephalus and Ophisurus serpens. HbF-Monserrato-Sassari [Gγ93(F9) Cys Arg] is an abnormal fetal hemoglobin observed in an apparently normal newborn baby during an hemoglobinopathies survey at birth in North Sardinian population. Sequencing of the γ globin genes revealed the TGT CGT transition at codon 93 in one of the two Gγ genes which causes the Arg for Cys amino acid replacement at position 93. The substitution was confirmed by mass spectrometric analysis which confirmed the presence of an additional Gγ chain whose mass value corresponds to the Arg Cys substitution already observed at the DNA level. Since modifications or substitutions at position β93 of HbA are known to affect the arrangement of a salt bridge at the α1β2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the HbF-Monserrato-Sassari variant were analyzed to evaluate the effect of the substitution at the same position of the Gγ chain. The data indicate that substitution Cys Arg causes a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity, with respect of normal HbF, indicating that Cys γ93 plays a key role for subunit cooperativity in the T R transition of the HbF tetramer. Since studies had shown that nitric oxide, the most important vasodilator agent, in the erytrocytes interacts in part with Cys β93 residues as S-nitrosohemoglobin (SNO-Hb), this variant, because of the loss of Cys γ93 residue, is considered to be potentially compromised in nitric oxide transport. Hb Roma [β115(G17)Ala Val] is an abnormal adult hemoglobin found in a 33- year-hold woman of Central Italian descent. We studied this previously uncharacterised variant because the β115(G17)Ala Val replacement occurs at the α1β1 (or α2β2) interface of the Hb molecule and then provides the opportunity to study how β115(G17)Ala Val substitution could perturb the hemoglobin structure and alter its function. Sequencing of the β-globin gene revealed the GCC GTC transition at codon 115 which causes the substitution of Ala, normally present at position β115, with Val. The substitution was confirmed by mass spectrometric analysis of the globins and tryptic peptides. The oxygen-binding properties, carried out on the mixture HbA/Hb Roma as the variant tetramer is undistinguishable by usual separation procedures, showed a significant increase in oxygen affinity with respect to normal HbA, both in the absence and presence of 2,3- bisphosphoglycerate. With regard to the two species of fish, Mugil cephalus and Ophisurus serpens, it is known that among the vertebrates, fish display the most extensive presence of multiple Hb components, which show considerable differences in amino acid sequence. These structural differences are often responsible for different functional properties. In the case of Mugil cephalus structural and functional characterisations of its hemoglobin system showed two major hemoglobins: HbI and HbII, which have the same functional properties, while hemoglobin system of O. serpens showed at least four anodic (isoelectric points, pI < 8) and one cathodic (pI > 8) components, which have different functional properties: the cathodic Hb may safeguard oxygen uptake under hypoxic and acidotic conditions, the anodic Hbs may be involved in pumping O2 in the swim bladder in order to maintain the neutral buoyancy of the fish. The natural hemoglobin variants are very important to evaluate how an amino acid replacement could perturb the hemoglobin molecule and alter its function, whereas the comparative analysis between hemoglobins of different species allows to verify how this protein might have changed during evolution in relation with the different physiological and habitat needs.