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Comprehensive characterization of tense and relaxed quaternary state glutaraldehyde polymerized bovine hemoglobin as a function of cross‐link density
Author(s) -
Gu Xiangming,
BoldenRush Crystal,
Cuddington Clayton T.,
Belcher Donald A.,
Savla Chintan,
Pires Ivan S.,
Palmer Andre F.
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27382
Subject(s) - diafiltration , glutaraldehyde , chemistry , methemoglobin , sodium dithionite , cooperativity , dispersity , hemoglobin , reaction rate constant , kinetics , cyanate , protein quaternary structure , chromatography , analytical chemistry (journal) , polymer chemistry , organic chemistry , biochemistry , membrane , physics , quantum mechanics , microfiltration , protein subunit , gene
Abstract Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this study, 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., O 2 affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand‐binding kinetics, auto‐oxidation, and haptoglobin [Hp]‐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 hemoglobin (bHb) molar ratio, gas‐liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T‐ and R‐state PolybHbs. Our results showed that, the duration of the fast‐step auto‐oxidation phase of R‐state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T‐state PolybHbs exhibited significantly higher bimolecular rate constants for binding to Hp and unimolecular O 2 offloading rate constants compared to R‐state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.