Semisynthetic hemoglobin A: Reconstitution of functional tetramer from semisynthetic α-globin

Girish Sahni, Youngnan J. Cho, K. Subramonia Iyer, Shabbir A. Khan, Ramnath Seetharam, A. Seetharama Acharya

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Abstract

The optimal conditions for the semisynthesis of α-globin through Staphylococcus aureus V8 protease condensation of a synthetic fragment (α1-30) with the complementary apo fragment (α31-141) in the presence of structure-inducing organic cosolvents and the reconstitution of the functional tetramer from semisynthetic α-globin have been investigated. The protease-catalyzed ligation of the complementary apo fragments α1-30 and α31-141 proceeds with very high selectivity at pH 6.0 and 4°C in the presence of 1-propanol as the organic cosolvent. A 30% 1-propanol solution was optimal for the semisynthetic reaction, and the synthetic reaction attained an equilibrium (approximately 50%) in 72 h. The synthetic reaction proceeds smoothly over a wide pH range (pH 5-8). Besides, the semisynthetic system is flexible, and it also proceeded well if trifluoroethanol or 2-propanol was used instead of 1-propanol. However, glycerol, a versatile organic cosolvent used in all other proteosynthetic reactions reported in the literature, was not very efficient as an organic cosolvent in the present synthetic reaction. The semisynthetic α-globin prepared with 1-propanol as the organic cosolvent has been reconstituted into HbA. The semisynthetic HbA was then purified by CM-cellulose chromatography. The semisynthetic HbA is indistinguishable from native HbA, in terms of its structural and functional properties. The semisynthetic approach provides the flexibility in protein engineering studies for the incorporation of spectroscopic labels (13C- and/or 15N-labeled amino acids), noncoded amino acids, or unnatural bond functionalities, which at present is not possible with genetic approaches.

Original languageEnglish (US)
Pages (from-to)5456-5461
Number of pages6
JournalBiochemistry
Volume28
Issue number13
Publication statusPublished - 1989

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Sahni, G., Cho, Y. J., Subramonia Iyer, K., Khan, S. A., Seetharam, R., & Seetharama Acharya, A. (1989). Semisynthetic hemoglobin A: Reconstitution of functional tetramer from semisynthetic α-globin. Biochemistry, 28(13), 5456-5461.