1.7Å and 2.1Å X-ray structures of Hemoglobin E and Hemoglobin A₂, isolated from the blood samples of b-thalassemic patients

J. K. Dattagupta, Udayaditya Sen, Debi Choudhury and Jhimli Dasgupta

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India (jiban@cmb2.saha.ernet.in)

Hemoglobin A₂ (a₂d₂), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent [1] and its elevated concentration in b-thalassemia is a useful clinical diagnostic. In b-thalassemia major, where there is a failure of b-chain production, HbA₂ acts as the predominant oxygen deliverer. Hemoglobin E, another common abnormal hemoglobin caused by splice site mutation in exon 1 of b globin gene, when combined with b-thalassemia causes severe microcytic anemia. The purification, crystallization and the structural studies of HbA₂ and HbE are reported here. HbA₂ and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from b-thalassemia minor and Eb-thalassemia. X-ray diffraction data of HbA₂ and HbE were collected upto 2.1Å and 1.73Å respectively. HbA₂ crystallized in space group P2₁ with unit cell parameters a=54.33Å, b=83.73Å, c=62.87Å, b=99.80° whereas HbE crystallized in space group P2₁2₁2₁ with unit cell parameters a=60.89Å, b=95.81Å, c=99.08Å [2]. Asymmetric unit in each case contains one Hb tetramer in R₂ state. The structure of HbA₂ and HbE were refined to an R factor of 18.3% (R_free 21.9%; PDB CODE 1NX5) and 19.1% (R_free 21.1%; PDB CODE 1NQP) respectively.

References

1 Nagel, R. L., Bookchin, R. M., Johnson, J., Labie, D., Wajcman, H., Isaac-Sodeye, W. A., Honig, G. R., Schiliro, G., Crookston, J. H., Matsutomo, K. (1979) Proc. Natl. Acad. Sci. 76(2), 670-672.

2 Dasgupta, J., Sen, U., Choudhury, D., Datta, P., Chakrabarti, A., Basu Chakrabarty, S., Chakrabarty, A. and Dattagupta, J. K. (2003) Biochem Biophys Res Commun. 303, 619-623.