A chromatographic study of the reaction sequence and effect of ligand on the reaction of human hemoglobin with negatively charged isothiocyanates: characterization of an intermediate modified only on the amino termini of thealpha chains

A HPLC investigation of the reaction of 4-isothiocyanatobenzoic acid and 4- isothiocyanatobenzenesulfonic acid with oxy- and deoxyhemoglobin was carrie d out. The initial reaction of aromatic isothiocyanato sulfonates and benzo ates with either oxy- or deoxyhemoglobin is with the amino termini of the a lpha chains followed by a much slower reaction with the amino termini of th e beta chains. Both reactions are much faster with deoxyhemoglobin than wit h oxyhemoglobin. An intermediate reacted only at the termini of the alpha c hains with 4-isothiocyanatobenzoic acid was isolated and purified and its f unctional properties determined. The intermediate showed a reduced oxygen a ffinity over a wide pH range and a reduced alkaline Bohr effect in the abse nce of chloride. The oxygen affinity of the intermediate showed a reduced h ut still significant response to chloride. (C) 2000 Elsevier Science B.V. A ll rights reserved.