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Comprehensive Characterization of Tense and Relaxed State Glutaraldehyde Polymerized Bovine Hemoglobin as a Function of Cross-link Density
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  • Xiangming Gu,
  • Clayton Cuddington,
  • Donald Belcher,
  • Chintan Savla,
  • Ivan Pires,
  • Andre Palmer
Xiangming Gu
The Ohio State University
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Clayton Cuddington
Ohio State University
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Donald Belcher
Ohio State University
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Chintan Savla
The Ohio State University
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Ivan Pires
Ohio State University College of Engineering
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Andre Palmer
The Ohio State University
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Abstract

Previously, our lab developed high molecular weight (MW) tense (T) state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this work, we prepared PolybHb in the T- and relaxed (R) quaternary state with ultrahigh MW (> 500 kDa) with varying cross-link densities and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, autoxidation, and haptoglobin-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e. cross-linker:bovine Hb (bHb) molar ratio, gas/liquid exchange time, temperature during dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T-state and R-state PolybHbs. Our results showed that, the duration of the fast-step autoxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHb exhibited significantly higher biomolecular rate constants for binding to haptoglobin and unimoleular O2 offloading rate constants compared to R-state PolybHb. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHb. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.

Peer review status:UNDER REVIEW

30 Mar 2020Submitted to Biotechnology and Bioengineering
30 Mar 2020Submission Checks Completed
30 Mar 2020Assigned to Editor
05 Apr 2020Reviewer(s) Assigned
26 Apr 2020Review(s) Completed, Editorial Evaluation Pending
26 Apr 2020Editorial Decision: Revise Major
07 May 20201st Revision Received
08 May 2020Submission Checks Completed
08 May 2020Assigned to Editor
09 May 2020Editorial Decision: Accept
09 May 2020Review(s) Completed, Editorial Evaluation Pending