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The equilibrium between mammalian hemoglobins and oxygen has long been recognized to be a reflection of functional interactions within individual az32 tetramers. The physical basis for such interactions has been the subject of many theoretical analyses, most of which postulate the existence of at least two forms of the protein with different reactivities toward the binding of oxygen.I-10 Any hypothesis attempting to relate structure and function must also account for such diverse phenomena as the pK of certain amino acid side chains, the rate of digestion of the protein with carboxypeptidase A, the dissociation of the tetramer into subunits, and the particular crystalline form of the protein."1-'4 These properties seem to be intimately associated with the binding of oxygen to hemoglobin, so that they behave as "linked functions."'15 16 We have approached the study of the cooperative interactions which give rise to the characteristic oxygen equilibrium of hemoglobin, and to those properties to which it is linked, by attempting to impose a constraint on the conformation of the protein. We hoped to achieve this by introducing covalent bridges in the hemoglobin molecule with a bifunctional reagent. Studies on the reaction of substituted maleimides with native human hemoglobin have demonstrated that the sulfhydryl group at position 93 in the ,3 chain reacts faster than any other residue in the molecule."7-'9 The second most reactive site is the N-terminal a-amino group of the a chain.'7 Still other residues may react with maleimides, although no other products have been characterized as yet. 18 20 Considerations of the selectivity exhibited by N-ethylmaleimide (NEM) in its reaction with hemoglobin, as well as the potential reactivity of a bifunctional maleimide derivative, led to the choice of bis(Nmaleimidomethyl)ether (BME) as a reagent to introduce covalent bridges into human and horse hemoglobin. We will present evidence that the conformational transition normally accompanying ligand exchange has been eliminated in these modified hemoglobins, and that BME hemoglobin assumes a deoxyhemoglobin-like conformation, even with the ligand attached. The physical and functional properties of these derivatives are consistent with the hypothesis that the normal conformation transition is intimately linked with cooperative interactions. Experimental.-Bis(N-maleimidomethyl)ether (methyl-C'4) was synthesized according to the method of Tawney et al.,2' by reacting maleimide with C'4-formaldehyde to form the methylol derivative, and, by elimination of water, condensing two molecules of N-methylolmaleimide to yield the final product. BME was reacted for 24 hr at 4VC with a 10% human or horse carbonmonoxyhemoglobin (COhemoglobin) solution, prepared from washed pooled erythrocytes by the method of Drabkin;22 the BAIE: hemoglobiti tetramer ratio was 2:1. Separation of products was achieved with tihe column chromatographic system of Clegg and Schroeder,23 using a 4 X 50-cm column of BioRex 70 and a discontinuous sodium phosphate gradient from 0.05 M to 0.2 M in Na+, all at pH 6.80. Peaks were analyzed for radioactive content by counting aliqliots on planchets. Ultracentrifugation studies were performed in double sector cells in the Spinco model E, using schlieren optics. Hemoglobin solutions (0.5%) were run at 40,000 rpm, and data were evaluated according to the methods described by Kirshner and Tanford,24 and by Kawahara

Chemical modification of hemoglobins: a study of conformation restraint by internal bridging.