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Affinity reagents for cross-linking hemoglobin: bis(phenoxycarbonylethyl)phosphinic acid (BPCEP) and
S P Peri1, V S Bhadti, K S Somerville-Armstrong
1Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore 21250, USA.
Abstract:
Synthesis and properties of two new macrobiomolecular cross-linking reagents, bis(phenoxycarbonylethyl) phosphinic acid (BPCEP) and bis(3-nitrophenoxycarbonylethyl)phosphinic acid (BNCEP), have been reported. The reagents were successfully employed to cross-link human hemoglobin under oxygenated conditions. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and fast protein liquid chromatography (FPLC) analyses of the reaction products indicated that the cross-link was intramolecular in nature, and that it was between the two beta subunits of hemoglobin in each case. The products were purified by DEAE-cellulose chromatography, and the purified material was employed for oxygen-binding assessments. The oxygen equilibrium curve of the cross-linked material, in each case, was right-shifted toward lower oxygen affinity as desired. The sigmoidal shapes of oxygen curves, in each case, suggested retainment of oxygen-binding cooperativity, although considerably lower than that of the native hemoglobin
