Proteosynthetic activity of immobilized Staphylococcus aureus V8 protease: Application in the semisynthesis of molecular variants of α-globin

Girish Sahni, A. Krishna Mallia, A. Seetharama Acharya

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

The proteosynthetic activity of Staphylococcus aureus V8 protease (endoproteinase Glu-C) immobilized onto cross-linked agarose beads by reductive alkylation procedure has been investigated. The overall substrate specificity of the enzyme, as judged by peptide mapping of performic acid oxidized RNase A, as well as the high propensity of the protease to slice selectively the α-chain of hemoglobin (Hb) A at the Glu(30)-Arg(31) peptide bond at pH 4.0 and 37°C was essentially unperturbed by the immobilization process. This high susceptibility of Glu(30) of the α-chain for proteolysis appears to be a consequence of the conformational aspects of the polypeptide in this region. The proteolysis of two mutant forms of α-chain, namely, those of Hb I (K16E) and Hb Sealy (D47H) by immobilized V8 protease at the Glu(30)-Arg(31) peptide bond proceeds with the same selectivity. The immobilized protease also retained the proteosynthetic activity, i.e., the ability to ligate the unprotected α-globin fragments at the Glu(30)-Arg(31) peptide bond in the presence of 30% 1-propanol. The use of the insoluble enzyme simplifies the procedures for the construction of new semisynthetic, molecular variants of α-globin. The general applicability of the immobilized enzyme for protein semisynthesis has been demonstrated by the construction of a doubly mutated α-globin. The complementary fragments from two natural mutant forms of α-globin, viz., α1-30 (K16E) from Hb I and α31-141 (D47H) from Hb Sealy, are readily ligated to form the double mutant α1-141 (K16E;D47H).

Original languageEnglish (US)
Pages (from-to)178-185
Number of pages8
JournalAnalytical Biochemistry
Volume193
Issue number2
DOIs
StatePublished - Mar 1991

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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