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Abstract The reaction of three N-substituted maleimides with horse oxy and deoxyhemoglobin has been studied. In all three cases, when the reactions were carried out with oxyhemoglobin, cooperativity between subunits was abolished (n = 1.0). When deoxyhemoglobin was used, cooperativity was reduced but not eliminated (n ∼ 2). A covalent bridge was introduced between cys F9(93)β (Perutz, M. F., J. Mol. Biol., 13, 646 (1965)) and his FG4(97)β when bis(N-maleimidomethyl)ether was reacted with horse oxyhemoglobin. When bis(N-maleimidomethyl)ether was reacted with deoxyhemoglobin, alkylation of one of the maleimide rings occurred at cys F9(93)β. The other maleimide ring did not react. Thus in contrast to oxyhemoglobin, no covalent bridge was formed in the protein. N-α-(bromoacetoxymethyl)maleimide (AM) reacted with horse oxyhemoglobin to link cys F9(93)β with val NA1(1)β'. This bridge decomposed rapidly leaving a carboxymethyl group on val NA1(1)β'. In the reaction of AM with deoxyhemoglobin, the ester bond in the reagent hydrolyzed after reaction with cys F9(93)β. Alkylation of the NH2 terminus of other β chains did not occur. N-α-(acetoxymethyl)maleimide, the monofunctional analogue of AM, reacted with both oxyhemoglobin and deoxyhemoglobin at cys F9(93)β. It thus appears that the structure and functional properties of the derivatives depend on the ligand state of the hemoglobin used in the reaction with the maleimides.
Arndt et al. (Thu,) studied this question.