The Interactions between α and β Chains of Human Hemoglobin

This paper describes experiments designed to show the linkage of the two chains in the αβ dimer. The approach has been a kinetic one. If modification of one chain leads to conformational change in the other, this should be detectable as a difference in the reaction velocities of functional groups on the unsubstituted chain. We studied three derivatives of human hemoglobin; one was modified in the α chain and two in the β chain. Substitution at the NH2 terminus of the α chain with 1-fluoro-2,4-dinitrobenzene leads to a total loss of cooperative interactions as judged by oxygenation parameters. In this derivative, the —SH group of cysteine 93β reacts more rapidly with N-ethylmaleimide than does the —SH group of unmodified hemoglobin. The effect of deoxygenation upon the reactivity of the 93β —SH is abolished. There is no change in the reactivity of cysteine 93β with iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid). The COOH termini of the α and β chains of the dinitrophenyl-carbonmonoxyhemoglobin are digested more slowly with carboxypeptidase B and A, respectively. When the primary substitution is made on the β chain at cysteine 93, the effects upon the α chain are less marked and depend on the substituent: N-ethylmaleimide has more potent effects than iodoacetamide. The N-ethylmaleimide derivative has a partial loss of cooperative interactions, as determined by oxygenation properties, while the iodoacetamide derivative shows normal oxygen binding except for an increased oxygen affinity. N-ethylmaleimide substitution at —SH 93β allows faster digestion of the α chain COOH terminus, but alkylation with iodoacetamide has no effect. Neither modification alters the reactivity of the NH2 terminus of the α chain to 1-fluoro-2,4-dinitrobenzene. Intrachain effects are produced by both substituents: substitution with N-ethylmaleimide slows and with iodoacetamide speeds the digestion of the β chain by carboxypeptidase A. Since modification of either chain of the dimer changes the reactivity of widely separated groups, a general conformational change must occur. The degree of change in the kinetics of the groups tested correlates with the degree of disruption of functional properties. These experiments show the close interaction of the two chains in the αβ dimer and are further evidence that the αβ dimer is the fundamental unit of both structure and function.