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.
Kwansa
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HE
Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201, USA.
Young
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Arosio
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Razynska
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Bucci
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D000495:Allosteric Site; D000818:Animals; D001241:Aspirin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D010108:Oxyhemoglobins / Q000378:metabolism; D010449:Peptide Mapping; D020836:Protein Structure, Quaternary; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D006454:Hemoglobins; D010108:Oxyhemoglobins; C036859:bis(3,5-dibromosalicyl)adipate; D002248:Carbon Monoxide; D014357:Trypsin; D001241:Aspirin; D010100:Oxygen