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HbS-Savaria: The Anti-polymerization Effect of a Single Mutation in Human α-chains

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Abstract

Recombinant α-Savaria globin (αS49R) was assembled with βS chains by the alloplex intermediate pathway to generate tetrameric rHbS-Sarvaria (α S49R2 β E6V2 ) that exhibited normal O2 affinity and co-operatively at pH 7.4. Allosteric effectors, 2,3-DPG, L35, and NaCl increased O2 affinity by 15%. Bohr effects were similar for rHbS-Savaria and HbS (0.38 ± 0.025 vs. 0.46 ± 0.03, respectively). The CSAT of HbS increased from 16.7 ± 0.8 to 27.0 ± 1.0 g/dL. Co-polymerization demonstrated inhibition predominantly by the Cis-dimer. Molecular modeling indicated that the positive charge at α-49 generated a strong anion-binding site and reduced flexibility of the CD-region by restricting movement in the E and F helices. The molecular distance between Arg-49 and Asn-78 in the neighboring double strand decreased, and electrostatic repulsion between the inter-double strands increased, resulting in inhibition of polymerization. The Savaria mutation may be useful for the design of super-inhibitory α-chains and gene therapy of sickle cell anemia.

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Abbreviations

βS :

Beta globin with HbS mutation

CSAT :

Concentration of deoxyHb in equilibrium with the polymer phase

2,3-DPG:

2,3-diphosphoglycerate

ESI-MS:

Electrospray ionization mass spectroscopy

HbS:

Hemoglobin S, sickle hemoglobin

IEF:

Isoelectric focusing

IHP:

Inositol hexaphosphate

L35:

2-[4-(3,5-dichlorophenylureido)phenoxy]-2-methylpropionic acid

LCR:

Locus control region

MEL:

Mouse erythroleukemia cells

p50:

The partial pressure of oxygen at half-saturation for Hb

RHbS-Savaria :

Recombinant HbS-Savaria

RP-HPLC:

Reverse phase high performance liquid chromatography

TFA:

Trifluoroacetic acid

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Acknowledgements

This work was supported by HL68962, HL55435, HL70994, and HL58512

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Authors and Affiliations

  1. Division of Hematology, Department of Medicine, Albert Einstein College of Medicine and Montefiore Hospital, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Sonati Srinivasulu, A. Seetharama Acharya, Muthuchidambaran Prabhakaran, Mary E. Fabry, Raouf Alami, Eric E. Bouhasirra & Ronald L. Nagel

  2. Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USA

    A. Seetharama Acharya & Ronald L. Nagel

  3. Microbiology Department, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA

    Steven N. Fiering

  4. Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA

    Eric E. Bouhasirra

Authors
  1. Sonati Srinivasulu
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  2. A. Seetharama Acharya
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  3. Muthuchidambaran Prabhakaran
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  4. Mary E. Fabry
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  7. Eric E. Bouhasirra
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  8. Ronald L. Nagel
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Correspondence to Ronald L. Nagel.

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Srinivasulu, S., Acharya, A.S., Prabhakaran, M. et al. HbS-Savaria: The Anti-polymerization Effect of a Single Mutation in Human α-chains . Protein J 26, 523–532 (2007). https://doi.org/10.1007/s10930-007-9089-9

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