The kinetics of assembly of normal and variant human oxyhemoglobins.
The kinetics of assembly have been monitored spectrophotometrically for normal and variant human oxyhemoglobins in 0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, at 21.5 degrees C. Oxyhemoglobin versus oxy chain static difference spectra were performed and revealed subtle but significant absorption changes in both the visible and Soret regions. Kinetic experiments were performed by rapidly mixing equivalent (in heme) concentrations of alpha and beta A chains and following the change in absorbance at 583 nm with time. Over a protein concentration range of 10-100 microM in heme prior to mixing, these time courses were homogeneous and followed first-order kinetics, yielding a value of 0.069 s-1 for the apparent rate constant of dissociation of oxygenated beta A chain tetramers. Under these conditions, the overall assembly of oxyhemoglobins S (beta 6Glu----Val) and N-Baltimore (beta 95Lys----Glu) were also governed by the rates of dissociation of their respective oxygenated beta S and beta N-Baltimore chain tetramers with the apparent first-order rate constants of 0.044 and 0.15 s-1, respectively. In the Soret region, the alpha, beta monomer combination reaction could be observed if the protein concentration (heme basis) was lowered and if protein nonequivalency (beta chain exceeded alpha chain concentration) mixing experiments were performed. A kinetic oxyhemoglobin A, oxy-alpha, oxy-beta A monomer difference spectrum could be generated, and simple second-order kinetics were observed (415 nm) yielding rate constants of 2.3, 3.3, and 4.8 X 10(5) M-1 s-1 for the assembly of oxyhemoglobins S, A, and N-Baltimore, respectively. To our knowledge, this is the first kinetic study to reveal significant differences between the rate of association of alpha and beta monomers of hemoglobin A and those of two distinctly charged hemoglobin variants.
D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D010108:Oxyhemoglobins / Q000378:metabolism*; D013053:Spectrophotometry
D046911:Macromolecular Substances; D010108:Oxyhemoglobins