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Magnetophoretic and Spectral Characterization of Oxyhemoglobin to Deoxyhemoglobin: Chemical vs Enzymatic Process
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  • Mitchell Weigand,
  • Jenifer Gómez-Pastora,
  • James Kim,
  • Matthew Kurek,
  • Richard Hickey,
  • David Irwin,
  • Maciej Zborowski,
  • Andre Palmer,
  • Jeffrey Chalmers
Mitchell Weigand
The Ohio State University
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Jenifer Gómez-Pastora
The Ohio State University
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James Kim
The Ohio State University
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Matthew Kurek
The Ohio State University
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Richard Hickey
Ohio State University College of Engineering
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David Irwin
University of Colorado - Anschutz Medical Campus
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Maciej Zborowski
Cleveland Clinic
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Andre Palmer
the Ohio State University
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Jeffrey Chalmers
The Ohio State University
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Abstract

A new method for hemoglobin (Hb) deoxygenation and re-oxygenation, in suspension or within red blood cells, RBCs, is described using the commercial enzyme product, EC-Oxyrase®. This method using EC-Oxyrase has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin, eliminating the need of a sparging deoxygenation gas and airtight vessels, as well as easy re-oxygenation by washing and adding to a normal buffer with dissolved oxygen (DO). Spectra of deoxyHb and metHb from RBCs using three preparation methods: sodium dithionite, sodium nitrite and Oxyrase, show high purity of the deoxyHb using Oxyrase (with little to no methemoglobin or hemichrome production from side reactions). The deoxygenation action of Oxyrase follows first order reaction kinetics. Paramagnetic characteristics of intracellular hemoglobin in RBCs are compared using cell tracking velocimetry for healthy and sickle cell disease (SCD) donors and oxygen dissociation curves show that the function of healthy RBCs is unchanged after Oxyrase treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyhemoglobin, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to the existing methods.

Peer review status:UNDER REVIEW

14 Oct 2020Submitted to Biotechnology and Bioengineering
15 Oct 2020Assigned to Editor
15 Oct 2020Submission Checks Completed
18 Oct 2020Reviewer(s) Assigned
22 Nov 2020Review(s) Completed, Editorial Evaluation Pending