Novel manufacturing method for producing apohemoglobin and its biophysical properties
| Autor: | Donald A. Belcher, Colbert F. Miller, Abraham K. Badu-Tawiah, Paul W. Buehler, Andre F. Palmer, Jin Hyen Baek, Ivan S. Pires, Richard Hickey |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Circular dichroism Erythrocytes Globular protein Size-exclusion chromatography Bioengineering Heme 01 natural sciences Applied Microbiology and Biotechnology Hemoglobins 03 medical and health sciences chemistry.chemical_compound 010608 biotechnology Humans Protein Unfolding chemistry.chemical_classification Gel electrophoresis Hemichrome Chromatography Ligand 030104 developmental biology chemistry Hemoglobin Apoproteins Hydrophobic and Hydrophilic Interactions Oxidation-Reduction Chromatography Liquid Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 117:125-145 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.27193 |
| Popis: | Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme-binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with 99% purity from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, -80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse-phase chromatography indicated that the mass of the α and β subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O2 equilibrium curve, O2 dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb. |
| Databáze: | OpenAIRE |
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