Transfer of human alpha- to beta-hemoglobin via its chaperone protein: evidence for a new state

The alpha-hemoglobin-stabilizing protein (AHSP), a small protein of 102 amino acids, is synthesized in red blood cell precursors. It binds specifically to alpha-hemoglobin (alpha-Hb) subunits acting as a chaperone protein, preventing the formation of alpha-hemoglobin-cytotoxic precipitates. We have...

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Bibliographic Details
Published in:The Journal of biological chemistry 2004-08, Vol.279 (35), p.36530
Main Authors: Baudin-Creuza, Véronique, Vasseur-Godbillon, Corinne, Pato, Christine, Préhu, Claude, Wajcman, Henri, Marden, Michael C
Format: Article
Language:English
Subjects:
Blood Proteins - chemistry
Blood Proteins - metabolism
Carbon Monoxide - chemistry
Dimerization
DNA, Complementary - metabolism
Glutathione Transferase - metabolism
Hemoglobins - chemistry
Hemoglobins - metabolism
Humans
Kinetics
Ligands
Molecular Chaperones - chemistry
Molecular Chaperones - metabolism
Plasmids - metabolism
Protein Binding
Protein Conformation
Recombinant Fusion Proteins - chemistry
Recombinant Proteins - chemistry
Spectrometry, Fluorescence
Time Factors
Tryptophan - chemistry
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Summary:The alpha-hemoglobin-stabilizing protein (AHSP), a small protein of 102 amino acids, is synthesized in red blood cell precursors. It binds specifically to alpha-hemoglobin (alpha-Hb) subunits acting as a chaperone protein, preventing the formation of alpha-hemoglobin-cytotoxic precipitates. We have engineered recombinant AHSP in a pGEX vector to study the functional consequence of interaction between AHSP and alpha-Hb. By in vitro binding assays, we have isolated the complexes glutathione S-transferase-AHSP.alpha-Hb and AHSP.alpha-Hb. The latter assembles as a heterodimer based on size-exclusion chromatography. These complexes exhibited monophasic CO binding kinetics, as observed for isolated alpha- and beta-subunits of hemoglobin. However, the rate of CO (or oxygen) binding to alpha-hemoglobin bound to its chaperone is three times slower than that observed for isolated alpha-hemoglobin, demonstrating a form that is intermediate to the R- and T-hemoglobin states. The physiologically relevant replacement of the chaperone by beta-hemoglobin chains could be detected by both ligand binding kinetics and tryptophan fluorescence quenching.
ISSN:0021-9258
DOI:10.1074/jbc.M405389200