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Biotechnology and Applied Biochemistry (1999) 29, (165–184) (Printed in Great Britain)

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Mutational analysis of sickle haemoglobin (Hb) gelation

Xianfeng Li*, Juha-Pekka Himanen†, Jose JavierMartin de Llano‡, Julio Cesar Padovan§, BrianT. Chait§ and James M. Manning*¹

*Department of Biology, Mugar 414, Northeastern University,360 Huntington Avenue, Boston, MA 02115, U.S.A., †MemorialSloan Kettering Cancer Center, 1300 York Avenue, New York, NY10021, U.S.A., ‡Instituto de InvestigacionesCitologicas, Fundacion Valenciana de Investigaciones Biomedicas,Amadeo de Saboya, 4, 46010 Valencia, Spain, and §RockefellerUniversity, Laboratory for Mass Spectrometry and Gaseous IonChemistry, 1230 York Avenue, New York, NY 10021, U.S.A.

Abbreviations used: DPG, 2,3-diphosphoglycerate; ESI–MS, electrospray ionization MS; HbS, sickle Hb; IEF, isoelectric focusing; MALDI–TOF-MS, matrix-assisted laser desorption ionization–time-of-flight MS; TFA, trifluoroacetic acid.

¹ To whom correspondence should be addressed.

The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures. We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human - and b-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells. The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization. These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter-tetrameric contact sites in the deoxy-HbS aggregate. This article reviews our studies on a number of sickle Hb mutants, including polymerization-enhancing HbS mutants and polymerization-inhibiting HbS mutants.

Received 7 January 1999; accepted 12 January 1999

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