Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
KINETIC INTERMEDIATES IN HEMOGLOBIN ASSOCIATION, DISSOCIATION, AND CARBON MONOXIDE BINDING REACTIONS
Abstract
A light-scattering stopped-flow device was used to study the kinetics of human oxyhemoglobin tetramer-dimer association and dissociation. The association reaction was induced by a rapid pH jump from 10.8 to 6.0, 7.0, and 8.0 in the stopped-flow apparatus. A simple 2D (DBLARR) T model did not accurately represent the observed kinetics, but a two dimer model, 2D' (--->) T (DBLARR) 2D, was consistent with all experimental results. The association reaction was also run with inositol hexaphosphate (IHP) which is known to bind to hemoglobin tetramers and dimers. When two IHP binding sites per tetramer and one for each dimer were assumed, and the IHP binding considered much more rapid than association, the two dimer model was shown to simulate accurately the observed kinetics. For both sets of experiments the mechanism of association was shown to proceed from an alkaline dimer form to an acid dimer form through an intermediate tetramer species. The human oxyhemoglobin dissociation reaction induced by pH jump from 7.0 to 10.8 could be represented by a simple T (DBLARR) 2D model, but plots of the apparent reciprocal relaxation time vs. hemoglobin concentration are suggestive of an intermediate tetrameric species in the dissociation pathway. While light-scattering measurements did not show positive evidence for the intermediate, several alternative models for dissociation were proposed. Stable kinetic intermediates in the carbon monoxide binding reaction to deoxyhemoglobin were prepared by using nonsaturating amounts of CO and rapidly blocking unliganded sites by ferricyanide oxidation or nitric oxide before the CO equilibrates among the heme sites. Analysis of individual chain CO populations derived from azide binding kinetics (met-CO intermediates) and ESR spectroscopy (NO-CO intermediates) showed that at all stages of ligation, alpha chains had higher populations of CO than beta chains. A heterogeneous, random binding, two state model and a sequential binding, two state model both failed to represent the results. A random binding, three state model accurately represented the population results, as well as absorbance changes for CO binding at 430 nm and around the static isosbestic point, 424.1 nm.
Subject Area
Chemistry
Recommended Citation
ZAMIS, THOMAS MICHAEL, "KINETIC INTERMEDIATES IN HEMOGLOBIN ASSOCIATION, DISSOCIATION, AND CARBON MONOXIDE BINDING REACTIONS" (1982). ETD collection for University of Nebraska-Lincoln. AAI8306520.
https://digitalcommons.unl.edu/dissertations/AAI8306520