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.

Human Carboxyhemoglobin at 2.2 Å Resolution: Structure and Solvent Comparisons of R-State, R2-State and T-State Hemoglobins

“…The studies were undertaken to provide an Rstate structure for comparison with the T-state and Rstate structures of human hemoglobin that are chemically modified (e.g. Bucci et al, 1996) or are variants with site-directed mutations (e.g. Fronticelli et al, 1994;Pechik et al, 1996) o.…”

Cys-93-ββ-Succinimidophenyl Polyethylene Glycol 2000 Hemoglobin A

“…It is the first ␤ 93 ␤ 93 intramolecularly cross-bridged species reported to date. The previously described bisdibromosalicyl derivatives of aliphatic carboxylic acids and the bispyridoxal phosphates stabilize the tetrameric structure of HbA by cross-linking the amino groups within its central cavity (1,(5)(6)(7)(8)(9)(10). In contrast, included in the atomic model used to calculate the F calc structure factor magnitudes and phases.…”

“…Several intramolecular cross-bridging approaches have been developed over the years for the stabilization of Hb tetramer against dissociation into ␣␤ dimers (1,(5)(6)(7)(8)(9)(10). By and large, the currently available intramolecular cross-bridging reagents are targeted to the reactive functional groups within the central cavity of Hb in domains that interact with allosteric effectors.…”

“…In fact, some of the cross-linkers have been designed to mimic the interactions of the allosteric effectors. Their design strategy has focused on the complementarity between the stereochemistry of the tagging ends of the proposed bi-or tri-functional reagents and the reactive functional groups within the central cavity (1,(5)(6)(7)(8)(9)(10). In view of their location within the central cavity in the modified Hbs, these cross-links offer steric hindrance for interactions with the allosteric effectors compared with unmodified HbA.…”

Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers

“…Stabilized bovine or human tetrameric Hb was prepared by intramolecular crosslink of lysine residues at β-82 by the reagent bis(3,5-dibromosalicyl) fumarate [11] or bis(3,5-dibromosalicyl)sebacate [12,13] to produce non-dissociable tetrameric Hbs with a P 50 of 18 Torr and cooperativity of n=1.7 or a P 50 of 34 Torr and cooperativity of n=2.1, respectively. Polymerization of Hb bovine has been described [1].…”