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Protein Science (2003), 12:690-695.
Copyright © 2003 The Protein Society

Stable octameric structure of recombinant hemoglobin {alpha}2ß283 Gly->Cys

Christophe Fablet1, Michael C. Marden1, Brian N. Green2, Chien Ho3, Josée Pagnier1 and Véronique Baudin-Creuza1

1 INSERM U 473, 94276 Le Kremlin-Bicêtre Cedex, France
2 Micromass UK Ltd., Altrincham, Cheshire WA14 5RZ, UK
3 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-2683, USA

Reprint requests to: Véronique Baudin-Creuza, INSERM U 473 84, rue du Général Leclerc, 94276 Le Kremlin-Bicêtre Cedex, France; e-mail: baudin{at}kb.inserm.fr; fax: 33-1-4959-5662.

We have engineered a recombinant hemoglobin (rHb ßG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb ßG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric ßG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric ßG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer ßG83C was not modified after incubation with fresh plasma.

Keywords: Hemoglobin; disulfide bridge; oligomerization; octamer; blood substitute

Abbreviations: DTT, dithiothreitol • DCL-Hb, diaspirin cross-linked hemoglobin • GSH, reduced glutathione • ESI-MS, electrospray ionization mass spectrometry • Hb, hemoglobin • Hb A, natural human hemoglobin • Hb-CO, carbonmonoxyhemoglobin • MetHbCN, cyanmethemoglobin


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