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

Sahni, G; Cho, Y J; Iyer, K S; Khan, S A; Seetharam, R; Acharya, A S

doi:10.1021/bi00439a021

Title: Semisynthetic hemoglobin A: Reconstitution of functional tetramer from semisynthetic. alpha. -globin

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Abstract

The optimal conditions for the semisynthesis of {alpha}-globin through Staphylococcus aureus V8 protease condensation of a synthetic fragment ({alpha}{sub 1-30}) with the complementary apo fragment ({alpha}{sub 31-141}) in the presence of structure-inducing organic cosolvents and the reconstitution of the functional tetramer from semisynthetic {alpha}-globin have been investigated. The protease-catalyzed ligation of the complementary apo fragments {alpha}{sub 1-30} and {alpha}{sub 31-141} proceeds with very high selectivity at pH 6.0 and 4{degree}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 {alpha}-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 andmore »« less

Authors:

Sahni, G; Cho, Y J; Iyer, K S; Khan, S A; Seetharam, R; Acharya, A S ^[1]

Rockefeller Univ., New York, NY (USA) Yeshiva Univ., Bronx, NY (USA)

Publication Date:: Tue Jun 27 00:00:00 EDT 1989

OSTI Identifier:: 5030708

Resource Type:: Journal Article

Journal Name:: Biochemistry; (USA)

Additional Journal Information:: Journal Volume: 28:13; Journal ID: ISSN 0006-2960

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; HEMOGLOBIN; BIOSYNTHESIS; LABELLED COMPOUNDS; UPTAKE; BIOCHEMISTRY; CARBON 13; DICHROISM; GLOBIN; NITROGEN 15; PEPTIDE HYDROLASES; STAPHYLOCOCCUS; BACTERIA; CARBON ISOTOPES; CARBOXYLIC ACIDS; CHEMISTRY; ENZYMES; EVEN-ODD NUCLEI; GLOBINS; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HYDROLASES; ISOTOPES; LIGHT NUCLEI; MICROORGANISMS; NITROGEN ISOTOPES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PIGMENTS; PORPHYRINS; PROTEINS; STABLE ISOTOPES; SYNTHESIS; 550201* - Biochemistry- Tracer Techniques

Citation Formats


                    Sahni, G, Cho, Y J, Iyer, K S, Khan, S A, Seetharam, R, and Acharya, A S. Semisynthetic hemoglobin A: Reconstitution of functional tetramer from semisynthetic. alpha. -globin.  United States: N. p., 1989. 
        Web.  doi:10.1021/bi00439a021.

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                    Sahni, G, Cho, Y J, Iyer, K S, Khan, S A, Seetharam, R, & Acharya, A S. Semisynthetic hemoglobin A: Reconstitution of functional tetramer from semisynthetic. alpha. -globin.  United States.  https://doi.org/10.1021/bi00439a021

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                    Sahni, G, Cho, Y J, Iyer, K S, Khan, S A, Seetharam, R, and Acharya, A S. 1989.  
        "Semisynthetic hemoglobin A: Reconstitution of functional tetramer from semisynthetic. alpha. -globin".  United States.  https://doi.org/10.1021/bi00439a021.

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@article{osti_5030708,

  title        = {Semisynthetic hemoglobin A: Reconstitution of functional tetramer from semisynthetic. alpha. -globin},

  author       = {Sahni, G and Cho, Y J and Iyer, K S and Khan, S A and Seetharam, R and Acharya, A S},

  abstractNote = {The optimal conditions for the semisynthesis of {alpha}-globin through Staphylococcus aureus V8 protease condensation of a synthetic fragment ({alpha}{sub 1-30}) with the complementary apo fragment ({alpha}{sub 31-141}) in the presence of structure-inducing organic cosolvents and the reconstitution of the functional tetramer from semisynthetic {alpha}-globin have been investigated. The protease-catalyzed ligation of the complementary apo fragments {alpha}{sub 1-30} and {alpha}{sub 31-141} proceeds with very high selectivity at pH 6.0 and 4{degree}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 {alpha}-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 ({sup 13}C- and/or {sup 15}N-labeled amino acids), noncoded amino acids, or unnatural bond functionalities, which at present is not possible with genetic approaches.},

  doi          = {10.1021/bi00439a021},

  url          = {https://www.osti.gov/biblio/5030708},
  journal      = {Biochemistry; (USA)},

  issn         = {0006-2960},

  number       = ,

  volume       = 28:13,

  place        = {United States},

  year         = {Tue Jun 27 00:00:00 EDT 1989},

  month        = {Tue Jun 27 00:00:00 EDT 1989}

}

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