Two other variants affecting the β76(E20) position have been described. This site is directed towards the exterior of the molecule and is near to the entry of the central cavity located between the two β chains (Fig. 4). The first variant is Hb J-Chicago, in which the alanine is replaced by an aspartic acid. Its functional properties are similar to those of Hb A [5]. The second is Hb Calais, which was found in a patient presenting with a chronic cyanosis [6]. In this case the alanine residue was replaced by a proline, resulting in a normally stable Hb but with decreased oxygen binding properties. From studies performed on bovine Hb, residue β76(E20) that is a lysine in these animals, seems to be an additional chloride-dependent regulatory site partly responsible for the decreased oxygen affinity [7]. In Hb Harlequin, the change from an alanine to a valine, is not expected to grossly alter the oxygen binding properties.

 The chromatographic behavior of Hb Harlequin is somewhat surprising. In the reversed phase HPLC system that we use, the b chain of Hb Harlequin was eluted together with bA. This differs drastically from what is observed for the b chain of Hb La Desirade [β129(H7) Ala—>Val], in which an identical amino acid exchange occurs. In an arbitrary scale, where bA elutes at 10 and a at 20, the β chain of Hb La Desirade elutes within the 15.1-15.5 window [2]. In contrast, the expected increase in hydrophobicity was found when the abnormal tryptic peptide was isolated by reversed phase HPLC. A possible explanation could be that the region of the Hb molecule around position E20 contains several charged residues [Asp 73 (E17), His 77 (EF1) and Asp 79 (EF3)] that may shield the alanine as well as the valine. The behavior of Hb Harlequin during ion exchange HPLC is also surprising. It is likely that minimal changes in the geometry of this area may alter the distribution of charges at the surface, and thus, their interactions with the cation exchange phase.