ENTROPY-DRIVEN INTERMEDIATE STEPS OF OXYGENATION MAY REGULATE THE ALLOSTERIC BEHAVIOR OF HEMOGLOBIN

When the oxygen binding isotherms of human, bovine and fallow deer (Da ma-Dama) hemoglobins are measured at different temperatures either by optical or calorimetric techniques, analyses according to the Adair's formalism show that at least one of the intermediate steps of ligation has a positive enthalpy change, i.e., absorbs rather than emitting he at, indicating that it is entropy rather than enthalpy driven (Bucci, E., et al. 1991. Biochemistry. 30:3195-3199; Bucci, E., et al. 1993. B iochemistry. 32:3519-3526; Johnson, C., et al. 1992. Biochemistry. 31: 10074-10082; Johnson, C., et at. 1995. Biophys. Chem. 59:107-117). Thi s phenomenon is confirmed in systems in which the beta 82 lysines of h uman hemoglobin are covalently cross-linked by acylation with dicarbox ylic acids of increasing length, namely the fumaryl (four-carbon-long) , adipoyl (six-carbon-long), and sebacoyl (10-carbon-long) residues. C onsistently in all of the systems here reported, the enthalpy excursio ns are masked by compensatory entropy changes, which keep the free ene rgy of ligand binding constant for the first three steps of oxygenatio n. Furthermore, the cooperativity index and the overall oxygen affinit y seem to be correlated to the positive enthalpy excursions of the int ermediate steps of ligation, Fumaryl-Hb (hemoglobin cross-linked with a fumaryl residue, four carbons) with the lowest absorption of heat ha s the highest affinity and lowest cooperativity index. Adipoyl-Hb (hem oglobin cross-linked with an adipoyl residue, six carbons) has the hig hest absorption of heat and the highest cooperativity index. It appear s that nonuniform heat release by the intermediates of oxygenation is part of the allosteric phenomena in hemoglobin systems. There is not e nough information that would allow assigning these phenomena to the in terplay of the various conformations described for hemoglobin besides the classic T (Fermi et at. 1984. J. Mol. Biol. 175:159-174) and R (Sh anaan. 1983. J. Mol. Biol. 171:31-59), as listed at the end of the Dis cussion. The possibility cannot be excluded that entropy-driven steps characterize new conformational transitions still to be described.