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Functional comparison of hemoglobin purified by different methods and their biophysical implications
Jacob Elmer1, Paul W Buehler, Yiping Jia
1William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio, USA.
Biotechnology and Bioengineering
|January 15, 2010
Summary
Tangential flow filtration (TFF) purifies hemoglobin (Hb) from red blood cells (RBCs) effectively. TFF-purified Hb shows quality comparable to commercial preparations, suitable for Hb-based oxygen carrier synthesis.
Area of Science:
- Biochemistry
- Biotechnology
- Materials Science
Background:
- Hemoglobin (Hb) purification from red blood cells (RBCs) often involves harsh conditions that can damage Hb structure and function.
- Commercial Hb preparations may undergo extensive processing, potentially introducing structural alterations.
Purpose of the Study:
- To compare the quality of Hb purified by tangential flow filtration (TFF) with commercial Hb preparations.
- To evaluate TFF as a suitable method for producing high-quality Hb for potential use in Hb-based oxygen carriers.
Main Methods:
- Tangential flow filtration (TFF) for Hb purification from bovine and human RBCs.
- Characterization using SDS-PAGE, SEC, ESI-MS, oxidation rate measurements (k(ox)), and analysis of responses to physiological conditions (pH, [Cl(-)], [IHP], T).
- Assessment of ligand binding kinetics for O(2), NO, and CO.
Main Results:
- TFF-purified Hbs exhibited comparable biophysical properties to commercial Hbs.
- A small amount of catalase in TFF-purified Hb reduced autoxidation rates.
- Mass spectrometry suggested structural alterations in chromatographically purified Hbs, but not in TFF-purified Hbs.
- TFF yielded highly pure Hb suitable for Hb-based O(2) carrier synthesis.
Conclusions:
- Tangential flow filtration (TFF) is an effective and suitable method for purifying hemoglobin from red blood cells.
- TFF-purified Hb demonstrates quality equivalent to commercial preparations, with potential advantages in structural integrity.
- The process yields highly pure Hb, applicable for the synthesis of Hb-based oxygen carriers.
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