Adult and fetal haemoglobin J-Sardegna [alpha 50(CE8)His -> Asp]: functional and molecular modelling studies

Haemoglobin (Hb) J-Sardegna [a50(CE8)His!Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 °C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3- DPG. A signi®cant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 °C. Hence in INTRODUCTION Haemoglobin (Hb) J-Sardegna [a50(CE8)His!Asp] was ®rst described in 1968 [1] and better identi®ed in 1989 [2] in subjects from the island of Sardinia. Structural analysis showed an aspartic acid residue in place of the histidine residue at position 50 of the a-globin chain in the region of the polypeptide chain between the C and E helices (CE8). The observed substitution is at the level of an external non-helical segment that, although not directly involved in the haem contacts [3,4], might contribute signi®cantly to maintaining the structure of the haem pocket and therefore to establishing the oxygen-binding properties of the molecule. The presence of Hb J-Sardegna does not seem to produce any particular clinical effect since appreciable clinical complaints have never been reported in heterozygous carriers of this abnormal Hb [5]. A newborn carrier of this mutation gave us the opportunity to study the functional properties of both fetal and adult Hb J-Sardegna (HbJ), which have never been investigated before. This system, therefore, offers the rare opportunity for characterization of a new Hb variant together with its fetal counterpart. Of course, since natural Hb variants have been and still are of great help for the elucidation of the critical role played by some amino acid residues, the interest in this system is also linked to the possibility of going deeper into the problem of structure± function interrelationships in a protein molecule. HbJ and its fetal form provide an excellent case for this type of investigation because the substitution of histidine by an aspartic acid residue will result in the loss of the interaction with Glu-a30 of the same Abbreviations used: Hb, haemoglobin; HbA, adult haemoglobin; HbF, fetal haemoglobin; HbJ, adult haemoglobin J-Sardegna; HbFJ, fetal haemoglobin J-Sardegna; 2,3-DPG, 2,3-diphosphoglycerate. 1 To whom correspondence should be addressed (e-mail b.giardina!uniserv.ccr.rm.cnr.it). fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the a"±b" interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [a50(CE8)His!Arg] at the same position.