Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients

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

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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).

Original languageEnglish
Pages (from-to)139-147
Number of pages9
JournalJournal of Biochemical and Biophysical Methods
Issue number3
Publication statusPublished - 1987


  • Hemoglobin valence intermediates
  • Immobilized pH gradients
  • Isoelectric focusing
  • Preparative electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics


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