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 acid (BCCEP, 1) have been reported. The reagent was designed with the aid of molecular modeling, employing crystal coordinates of human hemoglobin A0. 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 beta 1-beta 2 subunits. The oxygen equilibrium measurements of the modified hemoglobin at 37 degrees C showed significantly reduced oxygen affinity (P50 = 31.3 Torr) as compared with that of cell-free hemoglobin (P50 = 6.6 Torr). The sigmoidal shape of O2 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-HbA0 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.
Department of Chemistry and Biochemistry, University of Maryland, Baltimore 21250, USA.
R S
RS
Hosmane
S P
SP
Peri
V S
VS
Bhadti
V W
VW
Macdonald
D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D062385:Hydroxybenzoates / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D010721:Phosphinic Acids / Q000138:chemical synthesis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003432:Cross-Linking Reagents; D006454:Hemoglobins; D062385:Hydroxybenzoates; D010721:Phosphinic Acids; C109938:bis(2-(4-carboxyphenoxy)carbonylethyl) phosphinic acid