Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin.

Abstract

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 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 present at the second step of oxygenation, which has an affinity 27 times 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 with an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates 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, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31-59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation.