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

Journal Article · Tue Jun 27 00:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00439a021· OSTI ID:5030708

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

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

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.

• OSTI ID: 5030708
• Journal Information: Biochemistry; (USA), Vol. 28:13; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

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