Document Type

Project

Publication Date

Spring 6-2018

Keywords

Hemoglobin, Hemoglobin-based oxygen carrier

Abstract

Our long-term goal is to develop a monodisperse high molecular weight hemoglobin-based oxygen carrier (HBOC) for clinical care. One short-term aim is to ligate hemoglobin (Hb) molecules to a dendritic scaffold utilizing “click-chemistry”. Towards this goal, we have genetically modified the C-terminus of one of the α subunits of a di-α globin to contain the S. aureus sortase A recognition sequence (LPETG) and we have expressed the modified globin in E. coli. Here, we demonstrate that these Hbs can be site-specifically functionalized through sortase-mediated ligation of peptides containing dibenzocyclooctyne (DBCO). We further demonstrated proof-of-concept by conjugating an azide-funtionalized peptide with a fluorescent tag, 6-carboxyfluorescein (6-FAM) to Hb(DBCO). Additionally, work has been done to crystallize Hb with a site-specific mutant N108K, which promotes T-state stability of Hb (low oxygen affinity). Cell-free Hb is known to have high oxygen affinity and by determining the structural basis for improving T-state stability in our novel Hbs we hope to design our HBOC with ideal characteristics for reversible oxygen binding. This work establishes that we can functionalize hemoglobin with “click-chemistry” groups such as cyclooctyne, and conjugate that group to an azide with the ultimate goal of decorating azide-functionalized dendrimers with hemoglobin molecules to transport and exchange oxygen in the body.

Department

Chemistry

Subjects - Topical (LCSH)

Oxygen--Physiological transport; Blood substitutes;

Genre/Form

student projects; term papers

Type

Text

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 document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

Rights Statement

http://rightsstatements.org/vocab/InC/1.0/

Language

English

Format

application/pdf

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