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 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.
Baudin-Creuza
Véronique
V
INSERM U473, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France.
Vasseur-Godbillon
Corinne
C
Pato
Christine
C
Préhu
Claude
C
Wajcman
Henri
H
Marden
Michael C
MC
D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D005982:Glutathione Transferase / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
C468768:AHSP protein, human; D001798:Blood Proteins; D018076:DNA, Complementary; D006454:Hemoglobins; D008024:Ligands; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D002248:Carbon Monoxide; D014364:Tryptophan; D005982:Glutathione Transferase