A novel site-directed affinity reagent for cross-linking human hemoglobin: Bis[2-(4-phosphonooxyphenoxy)carbonylethyl]phosphinic acid

Bis[2-(4-phosphonooxyphenoxy)carbonylethyl]phosphinc acid (BPPCEP) was prepared and evaluated as a site-directed affinity reagent for cross-linking human hemoglobin. It was synthesized in four steps starting from 4-benzyloxyphenol and was converted to its pentasodium salt so as to afford efficient cross-linking in an aqueous medium. The reagent was found to specifically cross-link human hemoglobin A(0) in the beta-cleft chains under oxygenated reaction conditions at neutral pH. The amino acid residues involved in the cross-linking were determined by mass spectral analyses of tryptic digest fragments of cross-linked hemoglobin, employing a MALDI-TOF mass spectrometer. The MS analyses suggested that the most likely amino acids involved in the cross-links are Val-1 or Lys-82 present on one of the beta subunits and Lys-82 or Lys- 144 on the other. Molecular modeling studies performed on the reagent-HbA(0) complex corroborated the conclusions reached by MALDI-MS analyses. The oxygen equilibrium measurements of the three major BPPCEP-cross-linked Hb products, isolated and purified by preparative cation exchange chromatography, exhibited oxygen affinity (P-50) values of 14.5, 12.1, and 15.5 Torr as compared with the P50 of 13.1 Torr for cell-free hemoglobin. The oxygen-binding cooperativity of the modified products, as determined by the Hill coefficient generated from the Hill plots of the respective P-50 values, coupled with the absence of sigmoidal shape of the O-2 equilibrium curves, was considerably lower than that of the native hemoglobin.