POSITIVE AND NEGATIVE COOPERATIVITIES AT SUBSEQUENT STEPS OF OXYGENATION REGULATE THE ALLOSTERIC BEHAVIOR OF MULTISTATE SEBACYLHEMOGLOBIN

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3 ,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyse s confirm the presence of the 10 carbon atom long sebacyl residue cros s-linking the two beta 82 lysines of the beta-cleft (DecHb). The Adair 's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is p resent at the second step of oxygenation, which has an affinity 27 tim es lower than at the first step, Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity wit h an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 Angstrom resolution. The resulting atomic coordinat es are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol. Chem. 269, 23965-239 69] for deoxy-HbA. The electron density map of deoxy-DecHb indicates t he presence of the 10 carbon bridge between the beta 82 lysines. Molec ular modeling confirms that insertion of the linker into the T structu re requires only slight displacement of the two beta 82 lysines. Inste ad, insertion of the linker into the R and R2 structures [Shaanan (198 3) J. Mol. Biol. 171, 31-59; Silva et al, (1992) J. Biol. Chem. 267, 1 7248-17256] is hindered by serious sterical restrictions. The linker p rimarily affects the partially and fully liganded states of hemoglobin The data suggest in DecHb concerted conformational changes at each st ep of oxygenation.