Structure and function of Hb Saint-Jacques ( α2β2 140 (H18) Ala → Thr): A new high-oxygen-affinity variant with altered bisphosphoglycerate binding

Abstract

A low P 50 value in a fresh red blood cell suspension was discovered in a polycythemic patient (Hb 19 g·dl −1). Routine acid and alkaline electrophoreses of the hemolysate were identical to normal hemolysate. Isoelectrofocusing (pH gradient 6–8) did not reveal any abnormal band whether performed with the fully liganded or deoxygenated samples. Precise analyses of the oxygen dissociation curves of the propositus' red cells demonstrated a biphasic Hill plot, a normal Bohr effect and low interaction with 2,3-bisphosphoglycerate (2,3-DPG). Studies on the unfractionated hemolysate confirmed these observations and the inhibition of the effect of organic phosphates. Structural studies were carried out on the mixture of β A + β X chains and revealed the presence of two βTp14 peptides. Sequencing the abnormal βTp14 peptide showed the substitution Ala → Thr of the β140 (H18) residue. This new variant was named Hb Saint-Jacques. Examination of the three dimensional model of HbAo indicates that the substitution β140 (H18) Ala → Thr induces van der Waals interactions with the nearby lysine-82 (EF6) and leucine-81 (EF5) and a displacement of the EF corner of the β chains. This is likely to change the normal position of the lysine-82 (EF6), a major anionic binding site in the central cavity between the two β chains. Functional studies confirm the interpretation of a steric hindrance inhibiting the binding of large organic phosphates to Hb Saint-Jacques.