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Kinetic studies of the alkaline denaturation of human hemoglobin
Abstract
Kinetic studies of the alkaline denaturation of human oxy hemoglobin at pH 11.8 have shown that the process is more complicated than previously assumed. These studies employed stopped-flow diode array spectrophotometry (200-800 nm), a tandem NMR-optical stopped-flow device, and static circular dichroism (CD) measurements. When the heme concentration was reduced from 0.5 mM to 200 nM, the half-time decreased from about 5.5 min. to 3.8 sec. A model consistent with these absorbance data involved an equilibrium between the initial oxy dimer and an oxy monomer with the latter progressing further to at least two oxidized forms. The data were fit by a program that combined a stiff integrator and a Simplex minimization routine. At 2 mM in heme, a rapidly decaying intermediate was detected by NMR stopped-flow. Modelling the NMR data with the simultaneous optical changes allowed this intermediate to be assigned to the first oxidized monomer. For these NMR studies hemoglobin was specifically labelled at the $\beta$-93 cysteine with a $\sp{19}$F probe. The CD studies showed that about 40% alpha helix remained in the final state of various hemoglobin derivatives and that some residual structure remained in the vicinity of the heme. At pH 11.8 and 21$\sp\circ$C, the denaturation of human fetal hemoglobin is about one hundred times slower than that for adult hemoglobin. In terms of the kinetic model for the alkaline denaturation, the resistivity of fetal vs. adult hemoglobin derives from a much slower rate for the dissociation of the oxy dimer for fetal hemoglobin. The pH dependence of the denaturation implies the ionization of three residues in the dissociation of the $\alpha\sb1\beta\sb1$ dimer and is consistent with a general model proposed in 1970 by Prof. Max Perutz for the early stages of alkaline denaturation.
Subject Area
Biochemistry
Recommended Citation
McGee, William Andrew, "Kinetic studies of the alkaline denaturation of human hemoglobin" (1992). ETD collection for University of Nebraska-Lincoln. AAI9219378.
https://digitalcommons.unl.edu/dissertations/AAI9219378