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- Hajizadeh, Solmaz, et al.
(författare)
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Composite imprinted macroporous hydrogels for haemoglobin purification from cell homogenate
- 2018
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1534, s. 22-31
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Tidskriftsartikel (refereegranskat)abstract
- Purification of haemoglobin (Hb) has been studied for many years due to its ability to act as an oxygen carrier and its possible use in urgent clinical treatment. In this study, different types of chromatography columns were developed for Hb purification. Two of them showed satisfactory results as affinity chromatography columns and were thus studied more extensively. The affinity adsorbents were prepared by molecular imprinting techniques. In the first case, Pickering emulsion polymerization was used to prepare affinity adsorbents based on molecular imprinting technology. The imprinted particles were immobilized via covalent bonds on the surface of cryogel, a macroporous hydrogel produced by free radical polymerization under sub-zero temperature. In the second case, the affinity sites for Hb were formed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The dynamic binding capacity of the composite cryogel with the immobilized particles and the directly imprinted acrylamide cryogel was found to be 5.2 mg/g and 3.6 mg/g, respectively. The affinity columns showed high selectivity towards Hb in spite of the presence of serum albumin as well as other interfering substances in non-clarified cell homogenates. The maximum capacity in batch mode, the fluid flow and other physical and chemical properties of these columns were investigated.
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| 2. |
- Kettisen, Karin, et al.
(författare)
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Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin
- 2018
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Ingår i: Redox Biology. - : Elsevier BV. - 2213-2317. ; 19, s. 218-225
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Tidskriftsartikel (refereegranskat)abstract
- Redox active cysteine residues including βCys93 are part of hemoglobin's “oxidation hotspot”. Irreversible oxidation of βCys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active γCys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of γCys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at α19 on the exposed surface of the α-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.
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