Adipyl crosslinked bovine hemoglobins as new models of allosteric systems.

As indicated by peptide analyses and mass spectrometry estimations, intramolecular crosslink with bis(3,5-dibromosalicyl)adipate of bovine hemoglobin results in the formation of two main components covalently bridged across the beta-cleft. In one component the crosslink joins the beta(1)V1-beta(2)K81 residues (XL-Peak-1), in the other the bridge is between the beta(1)K81-beta(2)K81 residues (XL-Peak-2). Both components are tetrameric with a mass near MW = 67 kDa as estimated by gel filtration, and a hydrodynamic radius near 3. 20 nm, estimated by dynamic light scattering. They have very low oxygen affinity with Pm near 100 mmHg (XL-Peak-1) and near 70 mmHg (XL-Peak-2) respectively at 37 degrees C, at neutral pH. The Bohr effect is almost absent in XL-Peak-1, while in XL-Peak-2 it is very near normal. Both systems show oxygen binding cooperativity with an index near n = 2.0. Flash photolysis kinetics of the recombination with CO could be resolved into a fast and a slow component. The amplitude of the fast rates were not concentration-dependent. The stopped-flow kinetics were autoaccelerating, consistent with their ligand-binding cooperativity. All rates were very similar to those of normal hemoglobin, suggesting that the oxy- rather than the deoxy-forms of the systems were affected by the crosslink. Proteins 2000;39:166-169. Copyright 2000 Wiley-Liss, Inc.