Bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic Acid (BCCEP): A Novel Affinity Reagent for the β-Cleft Modification of Human Hemoglobin

Abstract The design, synthesis, and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4 carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP, 1 ) have been reported. The reagent was designed with the aid of molecular modeling, employing crystal coordinates of human hemoglobin A 0 . It was synthesized in three steps commencing from 4- t -butoxycarbonylphenol. The tri-sodium salt of 1 was employed to cross-link human oxyHb. While SDS–PAGE analyses of the modified hemoglobin product pointed to the molecular mass range of 32 kDa, the HPLC analyse suggested that the cross-link had formed between the β 1 -β 2 subunits. The oxygen equilibrium measurements of the modified hemoglobin at 37 °C showed significantly reduced oxygen affinity (P 50 =31.3 Torr) as compared with that of cell-free hemoglobin (P 50 =6.6 Torr). The sigmoidal shape of O 2 curves of the modified Hb pointed to reasonable retainment of oxygen-binding cooperativity after the cross-link formation. Molecular dynamics simulation studies on the reagent-HbA 0 complex suggested that the most likely amino acid residues involved in the cross-linking are N-terminus Val-1 or Lys-82 on one of the-chains, and Lys-144 on the other. These predictions were consistent with the results of MALDI-MS analyses of the peptide fragments obtained from tryptic digestion of the cross-linked product.