Hemoglobin Rothschild (β37(C3)Trp → Arg): A high/low affinity hemoglobin mutant

Abstract

In hemoglobin Rothschild arginine replaces the normal tryptophan at β37(C3), at α 1 β 2 contact. Residue β37 is in close proximity to Argα92 (FG4). Substitution of Trp by Arg at β37 results in two positively charged Arg residues at FG4 and C3 facing each other, a situation that would destabilize the subunit constraints essential for the tetrameric integrity of the molecule and for the reduced ligand affinity of unliganded normal HB ‡ ‡ Abbreviations used: Hb, hemoglobin; HbR, hemoglobin Rothschild; 2,3-DPG, 2.3-diphosphoglyceric acid. compared to isolated chains. Our studies show liganded HbR is extensively dissociated into dimers and has a high ligand affinity in phosphate buffer and a low ligand affinity in bis-Tris at alkaline pH. Kinetic studies indicate that in the T state HbR has a higher ligand affinity than HbA. This is explained by reduced subunit constraints in the T state and dissociation of the monoliganded species (Hb 4L) into dimers. Kinetic studies also show that R state Hb Rothschild has lower ligand affinity than R state HbA. These results are explained on the basis of extensive dissociation of R state Hb Rothschild into dimers and lower ligand affinity of dimers as compared to triliganded tetramers ( α 2 β 2(O 2) 3). Kinetic data indicate that the lower ligand affinity of dimers (Hb Rothschild) as compared to that of triliganded tetramers (HbA) is due to the increased ligand dissociation rates in the case of oxyhemoglobin and reduced ligand combination in the case of carboxyderivatives. Both the CO combination reaction time-course around 425 nm and the O 2 dissociation rates at 437.8 nm indicate the presence of large α,β-chain differences in Hb Rothschild.