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Date Permissions Signed

8-13-2018

Date of Award

Summer 2018

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

Antos, John M.

Abstract

Polymerized hemoglobin (poly-Hb) molecules have been shown to have reduced toxicity compared to cell-free hemoglobins when transfused intravenously. Poly-Hbs are typically generated with non-specific crosslinking agents that yield a product that is polydisperse in molecular weight. We propose a chemoenzymatic approach to generate poly-Hb of defined molecular weight. The proposed method employs the site-specific ligation reaction of the sortase A enzyme from S. aureus. A Hb mutant previously developed in our lab has been “sortagged” – modified by adding either the sortase recognition sequence (LPXTG) to the C-terminus of the α-subunit or a tetraglycine motif (GGGG) to the N-terminus. We show here that Hb subunits can be ligated directly by sortase A, using a mixture of substrate tagged (LPXTG) and nucleophile tagged (poly-G) Hbs. Additionally, using a novel approach for crosslinking Hb subunits using sortase A, we have generated a di-α construct that is functionalized with a strained cycloalkyne through an unnatural C-to-C fusion of sortagged α-subunits using a bifunctional sortase nucleophile peptide. We show that cycloalkyne-modified Hb molecules can be covalently linked to an azide functionalized fluorescein, using the well-established method of Huisgen cycloaddition. The work presented here establishes the feasibility of generating a monodisperse poly-Hb using azide decorated scaffolds.

Type

Text

Keywords

HBOCs, blood substitute, hemoglobin-based oxygen carriers, polymerized hemoglobins, poly-Hbs, RBC substitute, red blood cell substitute

DOI

https://doi.org/10.25710/gdsw-8274

Publisher

Western Washington University

OCLC Number

1049152445

Subject – LCSH

Hemoglobin--Synthesis; Blood--Transfusion

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this document 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.

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