Bis[2-(3-carboxyphenoxy)carbonylethyl]phosphinic acid (m-BCCEP): A novel affinity cross-linking reagent for the β-cleft modification of human hemoglobin

Hongyi Cai, Timothy A. Roach, Margaret Dabek, Karla S. Somerville, Seetharama Acharya, Ramachandra S. Hosmane

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1 Scopus citations


The design and synthesis of bis[2-(3-carboxyphenoxy)carbonylethyl] phosphinic acid (m-BCCEP, 1) as a site-directed affinity reagent for cross-linking human hemoglobin have been reported as part of our long-term goal to generate artificial blood for emergency transfusions. Molecular modeling techniques were used to design the reagent, employing crystal coordinates of human hemoglobin A0 imported from the Protein Data Bank. It was synthesized in four steps commencing from 3-hydroxybenzoic acid. The reagent 1 was converted to its trisodium salt to allow effective cross-linking in an aqueous medium. The reagent 1, as its trisodium salt, was found to specifically cross-link stroma-free human hemoglobin A0 in the β-cleft under oxygenated reaction conditions at neutral pH. The SDS-PAGE analyses of the modified hemoglobin pointed to the molecular mass range of 32 kDa as anticipated. The HPLC analyses of the product suggested that the cross-link had formed between the β12 subunits. Molecular dynamics simulation studies on the reagent-HbA0 complex suggested that the predominant amino acid residues involved in the cross-linking are N-terminus Val-1 or Lys-82 on one of the β-subunits and Lys-144 on the other. These predictions were borne out by MALDI-TOF MS analyses data of the peptide fragments obtained from tryptic digestion of the cross-linked product. The data also suggested the presence of a minor cross-link between Val-1 and Lys-82 on the opposing subunits. The oxygen equilibrium measurements of the m-BCCEP-modified hemoglobin product at 37 °C showed oxygen affinity (P 50 = 25.8 Torr) comparable to that of the natural whole blood (P 50 = 27.0 Torr) and significantly lower than that of stroma-free hemoglobin (P50 = 14.19 Torr) assayed under identical conditions. The measured Hill coefficient value of 1.91 of the m-BCCEP-modified Hb product points to the reasonable retainment of oxygen-binding cooperativity after the cross-link formation.

Original languageEnglish (US)
Pages (from-to)1494-1507
Number of pages14
JournalBioconjugate Chemistry
Issue number8
StatePublished - Aug 18 2010

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry


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