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Date Permissions Signed

2-17-2016

Date of Award

Winter 2016

Document Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Anthony-Cahill, Spencer J.

Second Advisor

Spiegel, P. Clint

Third Advisor

Murphy, Amanda R.

Abstract

Our research is focused on the production of a hemoglobin based oxygen carrier (HBOC) which can be used as a therapeutic in the event of acute blood loss. The administration of cell-free hemoglobin is associated with severe adverse effects due to dissociation of the tetrameric α₂β₂ complex into αβ heterodimers. Our approach to the design of an effective HBOC, is based on a recombinant circularly permuted human hemoglobin in which all of the subunits are linked in a single-chain fashion. This design would prevent the dissociation of the tetramer and allow for the biosynthesis of polymeric hemoglobins of defined mass. Preliminary ligand binding data with our permuted hemoglobins indicate that they prefer the high O₂-affinity R-state conformation over the low O₂-affinity T state. The βN108K and αV96W mutations were introduced to restore T state stability. Preliminary studies of the mutants have shown that while the βN108K mutation improved T-state stability, the αV96W mutation, in the context of the permuted hemoglobin backbone, displays an unexpected destabilizing effect on the T state. We have inserted the βN108K mutation into our single chain hemoglobin in order to obtain structural and functional data however it was discovered that this change significantly decreases expression yield. We would like to utilize X-ray crystallography to gain an atomic-level picture of protein structural differences that could explain the results from our αV96W mutants. Crystallization trials are underway for the αV96W mutant as well as the βN108K mutant and a αV96W + βN108K double mutant. To date no crystals have been obtained that diffract X-rays.

Type

Text

Publisher

Western Washington University

OCLC Number

942772711

Digital Format

application/pdf

Genre/Form

Academic theses

Language

English

Rights

Copying of this thesis in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this thesis for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.

Included in

Chemistry Commons

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