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KINETIC AND EQUILIBRIUM STUDIES ON THE ASSEMBLY OF THE BETA CHAINS OF HUMAN HEMOGLOBIN
Abstract
In order to study the association-dissociation kinetics of beta chains, analytical molecular sieve measurements were made to determine the stoichiometries and equilibrium constants. At pH 7.0 in phosphate buffer, the assembly of human (beta)CO, (beta)O(,2), deoxy (beta) and cyanomet (beta) chains can be presented as: tetramer-dimer-monomer. In contrast to human hemoglobin, liganded (beta) chains are less dissociated than deoxy (beta) chains. Rapid deoxygenation of (beta)O(,2) leads to increased dissociation of (beta) chains, while rapid CO binding to deoxy (beta) chains leads to increased association. Both light-scattering and absorbance changes are associated with these relaxation processes. Dual-beam light-scattering and absorbance stopped-flow devices were used to determine two dissociation rate constants, k(,42) and k(,21), for both (beta)CO and deoxy (beta) chains. The association rate constants, k(,24) and k(,12), were then obtained from the equilibrium constants. The k(,42) rate constants for (beta)CO and deoxy (beta) were very sensitive to phosphate concentration. An experiment in which deoxy (beta) chains at pH 9.0 were quickly converted to (beta)CO at pH 7.0 and the subsequent light-scattering changes followed, showed that the conformation of the (beta) dimer at pH 9.0 is different from that at pH 7.0. The rate of the conformational change appears to be slow (t(, 1/2)(TURNEQ)3 min). The reaction of p-mercurybenzoate with sulfhydryls on the (beta) chains is complex. The triphasic absorbance changes at 254 nm associated with PMB binding showed that both SH's on the (beta) monomer and one (or 2) on the dimer react rapidly with PMB. The observed light-scattering changes show that (beta)('PMB) chains dissociate in the course of PMB binding. These kinetic results allowed us to suggest a reasonable mechanism for PMB binding to (beta) chains. The self-assemblies of the isolated carp (alpha) and (beta) chains were studied by large-zone gel-filtration. Carp (beta)CO at pH 7.0 was found to be more dissociated than deoxy (beta). At pH 9.0, both (beta)CO and deoxy (beta) chains are tightly associated. Carp (alpha)CO and deoxy (alpha) chains show a D (DBLHARR) 2M stoichiometry, similar to human (alpha)CO at pH 7.0.
Subject Area
Chemistry
Recommended Citation
PHAM, THANG TAT, "KINETIC AND EQUILIBRIUM STUDIES ON THE ASSEMBLY OF THE BETA CHAINS OF HUMAN HEMOGLOBIN" (1981). ETD collection for University of Nebraska-Lincoln. AAI8208370.
https://digitalcommons.unl.edu/dissertations/AAI8208370