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Functional comparison of hemoglobin purified by different methods and their biophysical implications
Author(s) -
Elmer Jacob,
Buehler Paul W.,
Jia Yiping,
Wood Francine,
Harris David R.,
Alayash Abdu I.,
Palmer Andre F.
Publication year - 2010
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.22659
Subject(s) - chemistry , hemoglobin , chromatography , heme , yield (engineering) , autoxidation , size exclusion chromatography , recombinant dna , kinetics , biochemistry , enzyme , materials science , physics , quantum mechanics , metallurgy , gene
Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation ( k ox ), responses to physiological conditions (pH, [Cl − ], [IHP], and T ), and ligand binding kinetics (O 2 , NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O 2 carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85. © 2010 Wiley Periodicals, Inc.