Synthetic potential of Staphylococcus aureus V8‐protease: An approach toward semisynthesis of covalent analogs of α‐chain of hemoglobin S

Ramnath Seetharam, A. Seetharama Acharya

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Enzyme‐catalyzed reformation of peptide bonds in the noncovalent fragment systems of proteins has been emerging as a convenient procedure for the semisynthesis of covalent analogs of the respective proteins. Limited proteolysis of the α‐chain of hemoglobin S with Staphylococcus aureus V8‐protease converts the chain into a fragment‐complementing system by hydrolyzing the peptide bond Glu(30)‐Arg(31) of the chain. Therefore, it is conceivable that semisynthesis of covalent analogs of α‐chain could be achieved if conditions for the V8‐protease catalyzed formation of peptide bonds could be established. The synthetic potential of V8‐protease has been now investigated by incubating V8‐protease‐derived fragments of α‐chain, namely α1–30 and α31–47 with the enzyme at pH 6.0 in the presence of n‐propanol as the organic cosolvent. RP high performance liquid chromatography analysis showed that a new chromatographically distinct component is generated on incubation, and this has been identified as α1–47 by amino acid analysis, redigestion with V8‐protease (in the absence of n‐propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of α1–47 is at pH 6.0, 4°C, and 24 hr of incubation with 25% n‐propanol as organic cosolvent. This stereospecific condensation of the fragments proceeded to a high level of about 50% in 24 hr. Further incubation up to 72 hr did not increase the yield of α1–47, suggesting that an equilibration of synthesis and hydrolysis reactions has been attained. The demonstration of the synthetic potential of V8‐protease and the fact that α1–30 and α31–141 interact to form a native‐like complex, opens up an approach for the semisynthesis of covalent analogs of α‐chain of hemoglobin S.

Original languageEnglish (US)
Pages (from-to)87-99
Number of pages13
JournalJournal of Cellular Biochemistry
Volume30
Issue number1
DOIs
StatePublished - 1986
Externally publishedYes

Fingerprint

Sickle Hemoglobin
Staphylococcus aureus
Peptides
Peptide Mapping
Proteolysis
High performance liquid chromatography
Proteins
Hydrolysis
High Pressure Liquid Chromatography
Amino Acids
Condensation
Demonstrations
Enzymes

Keywords

  • gelation
  • semisynthesis
  • V8‐protease

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Synthetic potential of Staphylococcus aureus V8‐protease : An approach toward semisynthesis of covalent analogs of α‐chain of hemoglobin S. / Seetharam, Ramnath; Acharya, A. Seetharama.

In: Journal of Cellular Biochemistry, Vol. 30, No. 1, 1986, p. 87-99.

Research output: Contribution to journalArticle

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abstract = "Enzyme‐catalyzed reformation of peptide bonds in the noncovalent fragment systems of proteins has been emerging as a convenient procedure for the semisynthesis of covalent analogs of the respective proteins. Limited proteolysis of the α‐chain of hemoglobin S with Staphylococcus aureus V8‐protease converts the chain into a fragment‐complementing system by hydrolyzing the peptide bond Glu(30)‐Arg(31) of the chain. Therefore, it is conceivable that semisynthesis of covalent analogs of α‐chain could be achieved if conditions for the V8‐protease catalyzed formation of peptide bonds could be established. The synthetic potential of V8‐protease has been now investigated by incubating V8‐protease‐derived fragments of α‐chain, namely α1–30 and α31–47 with the enzyme at pH 6.0 in the presence of n‐propanol as the organic cosolvent. RP high performance liquid chromatography analysis showed that a new chromatographically distinct component is generated on incubation, and this has been identified as α1–47 by amino acid analysis, redigestion with V8‐protease (in the absence of n‐propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of α1–47 is at pH 6.0, 4°C, and 24 hr of incubation with 25{\%} n‐propanol as organic cosolvent. This stereospecific condensation of the fragments proceeded to a high level of about 50{\%} in 24 hr. Further incubation up to 72 hr did not increase the yield of α1–47, suggesting that an equilibration of synthesis and hydrolysis reactions has been attained. The demonstration of the synthetic potential of V8‐protease and the fact that α1–30 and α31–141 interact to form a native‐like complex, opens up an approach for the semisynthesis of covalent analogs of α‐chain of hemoglobin S.",
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