BIS[2-(4-CARBOXYPHENOXY)CARBONYLETHYL]PHOSPHINIC ACID (BCCEP) - A NOVEL AFFINITY REAGENT FOR THE BETA-CLEFT MODIFICATION OF HUMAN HEMOGLOBIN

The design, synthesis, and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4 carboxyphenoxy)carbonylethyl]phosphinic aci d (BCCEP, 1) have been reported. The reagent was designed with the aid of molecular modeling, employing crystal coordinates of human hemoglo bin Ao. It was synthesized in three steps commencing from 4-t-butoxyca rbonylphenol. The tri-sodium salt of 1 was employed to cross-link huma n oxyHb. While SDS-PAGE analyses of the modified hemoglobin product po inted to the molecular mass range of 32 kDa, the HPLC analyse suggeste d that the cross-link had formed between the beta(1)-beta(2) subunits. The oxygen equilibrium measurements of the modified hemoglobin at 37 degrees C showed significantly reduced oxygen affinity (P-50 = 3 1.3 T orr) 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 reason able retainment of oxygen-binding cooperativity after the cross-link f ormation. Molecular dynamics simulation studies on the reagent-HbA(o) 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 t ryptic digestion of the cross-linked product. (C) 1998 Elsevier Scienc e Ltd. All rights reserved.