Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli–responsive affinity macroligand

Title data

Stocker-Majd, Gisela ; Hilbrig, Frank ; Freitag, Ruth:
Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli–responsive affinity macroligand.
In: Journal of Chromatography A. Vol. 1194 (2008) Issue 1 . - pp. 57-65.
ISSN 0021-9673
DOI: https://doi.org/10.1016/j.chroma.2008.03.091

Official URL:

Abstract in another language

Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin–haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity.

Further data

Item Type:	Article in a journal
Refereed:	Yes
Additional notes:	27th International Symposium on the Separation of Proteins, Peptides & Polynucleotides
Keywords:	Thermoresponsive polymer
Institutions of the University:	Faculties > Faculty of Engineering Science > Chair Process Biotechnology > Chair Process Biotechnology - Univ.-Prof. Dr. Ruth Freitag Faculties Faculties > Faculty of Engineering Science Faculties > Faculty of Engineering Science > Chair Process Biotechnology
Result of work at the UBT:	No
DDC Subjects:	600 Technology, medicine, applied sciences 600 Technology, medicine, applied sciences > 620 Engineering
Date Deposited:	24 Feb 2016 14:25
Last Modified:	30 Jun 2022 10:19
URI:	https://eref.uni-bayreuth.de/id/eprint/31038