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

Abstract

The optimal conditions for the semisynthesis of alpha-globin through Staphylococcus aureus V8 protease condensation of a synthetic fragment (alpha 1-30) with the complementary apo fragment (alpha 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 1-30 and alpha 31-141 proceeds with very high selectivity at pH 6.0 and 4 degrees 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 (13C- and/or 15N-labeled amino acids), noncoded amino acids, or unnatural bond functionalities, which at present is not possible with genetic approaches.

Conformationally driven protease‐catalyzed splicing of peptide segments: V8 protease‐mediated synthesis of fragments derived from thermolysin and ribonuclease A

“…1-Propanol was chosen as a cosolvent based on earlier semisynthetic studies of aglobin (Sahni et al, 1989;Roy et al, 1992). Accordingly, the effect of the concentration of 1-propanol (cosolvent) on the conformation of TC-peptide was monitored by CD spectroscopy (Fig.…”

High-efficiency transpeptidation catalysed by clostripain and electrostatic effects in substrate specificity

“…Proteinases are efficient tools for peptide synthesis [1,2]. However, they have been applied to the fragment condensation of long-chain peptides only in limited cases, usually assisted by specific interactions between acyl and amine components [1][2][3][4][5]. the main reason for the limited application is the low solubility of long-chain peptides in terms of molar concentration.…”

Hemoglobin Einstein: Semisynthetic deletion in the B‐helix of the α‐chain

“…A discontinuity between Glu 30 and Arg 31 of the α‐chain is permissible within the tertiary interactions of the chain (Seetharam and Acharya 1986; Seetharam et al 1986). However, this discontinuity is not permissible within the constraints of the quaternary structure of Hb (Sahni et al 1989).…”

“…The unusually high selectivity of the Glu‐Arg peptide bond of the isolated α‐chain or α‐globin of Hb to V8 protease cleavage has been translated into a semisynthetic procedure (Sahni et al 1989, 1991; Roy and Acharya 1994), permitting the splicing of α 1–30 , either synthesized chemically or generated from the slicing of the animal α‐globin, with either human or animal α 31–141 to generate ss‐chimeric α‐globin (Roy et al 1993; Nacharaju et al 1997; Srinivasulu et al 1999; Rao et al 2000). The chimeric α‐globins readily assemble with human β A ‐ or β S ‐chains and generate functional tetramers containing chimeric α‐chains even when they carry a significant number of sequence differences in the amino‐terminal region of the B‐helix.…”