The Reaction of N-α-(Bromoacetoxymethyl)maleimide with Hemoglobin

Abstract The reaction of N-α-(bromoacetoxymethyl)maleimide (AM) with human and horse hemoglobin was observed over a range of pH and ligand conditions. In the case of human hemoglobin, cys F9(93)β (Perutz, M. F., J. Mol. Biol., 13, 646 (1965)) was alkylated by the maleimide ring. This was followed by rapid hydrolysis of the ester bond in the reagent. With horse oxyhemoglobin, a transient covalent bridge between β chains was formed by reaction of the maleimide ring with cys F9(93)β followed by reaction of the bromoacetyl portion of the reagent with val NA1(1)β of the other β chain in the tetramer. After hydrolysis of the ester bond, which occurred spontaneously, the resulting hemoglobin derivative showed no cooperative interactions. A comparison of the dissociation behavior and rates of carboxypeptidase A digestion of horse oxy- and deoxy-AM-hemoglobin showed that the derivative resembled the oxy form of horse hemoglobin even in the absence of ligands. The alkylation of horse oxyhemoglobin could be carried out in two steps. By first reacting the AM reagent with cyanomethemoglobin at pH 5.7, the cys F9(93)β sulfhydryl group was alkylated. Addition of excess carbonmonoxyhemoglobin and raising the pH to 7 resulted in alkylation of the NH2 termini of the β chains by the bromoacetyl portion of the reagent. Separation of these hemoglobin derivatives was achieved. The two-step reaction sequence was also used to examine one feature of the conformation of a mixed cyanomet-AM-deoxyhemoglobin tetramer.