Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phasse high-performance liquid chromatography

Rick J. Krueger, T. R. Hobbs, Kevin A. Mihal, J. Tehrani, M. G. Zeece

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

The specificity and rate of cleavage of adrenocorticotrophic hormone (ACTH) peptide bonds by endoproteinase Arg C were analyzed using capillary electrophoresis (CE) and reversed-phase (C18 high-performance liquid chromatography (HPLC). Acidic cleavage products were readily resolved by Ce in uncoated cpaillries using low ionic strength electrolytes. However, products predicted to have a net positive charge greater than 2 or more than 4 positively charged groups per peptide did not migrate out from the capillary at low ionic strength. Addition of salts and zwitterions to the electrolyte decreased capillary-peptide interactions such that all of the ACTH peptides examined were eluted with high efficiency separation by Ce. Commercially obtained endoproteinsane Arg C preparations exhibited peptidase activity at Lys15Lys16 and at Lys16Arg17 in additionto the expected cleavage at Argz.sbnd;X bonds. ACTH peptide bond cleavage rates for Arg18Trp9, Arg17Arg18, Lys15Lys16, adn Lys167z.sbnd;Arg17 were 1.46, 0.096, 0.057, and 0.029 μmol min-1 mg-1 respectively. CE separations generally exhibited better resolution and were accomplished in shorter times than C18 HPLC separations. These properties make CE a particularly appropriate method for kinetic analysis of proteolytic enzyme action on peptide substrates.

Original languageEnglish (US)
Pages (from-to)451-461
Number of pages11
JournalJournal of Chromatography A
Volume543
Issue numberC
DOIs
StatePublished - 1991

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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