Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients

Cecilia Gelfi; Pier Giorgio Righetti; Ermanna Rovida; Michele Samaja

doi:10.1016/0165-022X(87)90016-9

Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients

Cecilia Gelfi, Pier Giorgio Righetti, Ermanna Rovida, Michele Samaja

IRCCS Policlinico San Donato

Research output: Contribution to journal › Article

Abstract

We compare three separation techniques for preparative purposes, i.e. ion-exchange chromatography on CM-cellulose, conventional isoelectric focusing in polyacrylamide gel slabs and immobilized pH gradients. The biological system used to test the three methods is a solution containing four hemoglobin (Hb) valence intermediates, i.e. metHb, oxyHb, (α⁺β^O₂)₂ and (α^O₂β⁺)₂. The ΔpI between the two valence intermediates is 0.04 pH units. Immobilized pH gradients give the best performance in terms of resolving power, total amount of protein which can be loaded and retention of biological activity by the protein (the latter assessed by determination of CO dissociation rates).

Original language	English
Pages (from-to)	139-147
Number of pages	9
Journal	Journal of Biochemical and Biophysical Methods
Volume	14
Issue number	3
DOIs	https://doi.org/10.1016/0165-022X(87)90016-9
Publication status	Published - 1987

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Keywords

Hemoglobin valence intermediates
Immobilized pH gradients
Isoelectric focusing
Preparative electrophoresis

ASJC Scopus subject areas

Biochemistry
Biophysics

Cite this

Gelfi, C., Giorgio Righetti, P., Rovida, E., & Samaja, M. (1987). Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients. Journal of Biochemical and Biophysical Methods, 14(3), 139-147. https://doi.org/10.1016/0165-022X(87)90016-9

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AU - Gelfi, Cecilia

AU - Giorgio Righetti, Pier

AU - Rovida, Ermanna

AU - Samaja, Michele

PY - 1987

Y1 - 1987

N2 - We compare three separation techniques for preparative purposes, i.e. ion-exchange chromatography on CM-cellulose, conventional isoelectric focusing in polyacrylamide gel slabs and immobilized pH gradients. The biological system used to test the three methods is a solution containing four hemoglobin (Hb) valence intermediates, i.e. metHb, oxyHb, (α+βO2 )2 and (αO2 β+)2. The ΔpI between the two valence intermediates is 0.04 pH units. Immobilized pH gradients give the best performance in terms of resolving power, total amount of protein which can be loaded and retention of biological activity by the protein (the latter assessed by determination of CO dissociation rates).

AB - We compare three separation techniques for preparative purposes, i.e. ion-exchange chromatography on CM-cellulose, conventional isoelectric focusing in polyacrylamide gel slabs and immobilized pH gradients. The biological system used to test the three methods is a solution containing four hemoglobin (Hb) valence intermediates, i.e. metHb, oxyHb, (α+βO2 )2 and (αO2 β+)2. The ΔpI between the two valence intermediates is 0.04 pH units. Immobilized pH gradients give the best performance in terms of resolving power, total amount of protein which can be loaded and retention of biological activity by the protein (the latter assessed by determination of CO dissociation rates).

KW - Hemoglobin valence intermediates

KW - Immobilized pH gradients

KW - Isoelectric focusing

KW - Preparative electrophoresis

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10.1016/0165-022X(87)90016-9