title: Preparation and kinetic characterization of a series of betaW37 variants of human hemoglobin A: evidence for high-affinity T quaternary structures.
abstract: Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.
authors.affiliation: Department of Medicine, University at Buffalo Veterans Administration Medical Center, Buffalo, New York 14215, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Kwiatkowski
authors.forename: H L
authors.initials: HL
authors.lastname: Hui
authors.forename: A
authors.initials: A
authors.lastname: Wierzba
authors.forename: R W
authors.initials: RW
authors.lastname: Noble
authors.forename: R Y
authors.initials: RY
authors.lastname: Walder
authors.forename: E S
authors.initials: ES
authors.lastname: Peterson
authors.forename: S G
authors.initials: SG
authors.lastname: Sligar
authors.forename: K E
authors.initials: KE
authors.lastname: Sanders
mesh_terms: D019943:Amino Acid Substitution; D002248:Carbon Monoxide / Q000378:metabolism / Q000528:radiation effects; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005914:Globins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006441:Hemoglobin A / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D009154:Mutation; D010782:Photolysis; D010833:Phytic Acid / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D010833:Phytic Acid; D002248:Carbon Monoxide; D014364:Tryptophan; D005914:Globins; D006441:Hemoglobin A