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

27 Citations (Scopus)

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

Fingerprint

Capillary electrophoresis
Capillary Electrophoresis
High performance liquid chromatography
Adrenocorticotropic Hormone
High Pressure Liquid Chromatography
Peptides
Ionic strength
Osmolar Concentration
Electrolytes
Peptide Hydrolases
submandibular proteinase A
Salts
Kinetics
Substrates

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

Cite this

Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phasse high-performance liquid chromatography. / Krueger, Rick J.; Hobbs, T. R.; Mihal, Kevin A.; Tehrani, J.; Zeece, M. G.

In: Journal of Chromatography A, Vol. 543, No. C, 1991, p. 451-461.

Research output: Contribution to journalArticle

Krueger, Rick J. ; Hobbs, T. R. ; Mihal, Kevin A. ; Tehrani, J. ; Zeece, M. G. / Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phasse high-performance liquid chromatography. In: Journal of Chromatography A. 1991 ; Vol. 543, No. C. pp. 451-461.
@article{aa8a17bc87864a4fbba7dd9110cc58af,
title = "Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phasse high-performance liquid chromatography",
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.",
author = "Krueger, {Rick J.} and Hobbs, {T. R.} and Mihal, {Kevin A.} and J. Tehrani and Zeece, {M. G.}",
year = "1991",
doi = "10.1016/S0021-9673(01)95796-6",
language = "English (US)",
volume = "543",
pages = "451--461",
journal = "Journal of Chromatography A",
issn = "0021-9673",
number = "C",

}

TY - JOUR

T1 - Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phasse high-performance liquid chromatography

AU - Krueger, Rick J.

AU - Hobbs, T. R.

AU - Mihal, Kevin A.

AU - Tehrani, J.

AU - Zeece, M. G.

PY - 1991

Y1 - 1991

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0025849707&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025849707&partnerID=8YFLogxK

U2 - 10.1016/S0021-9673(01)95796-6

DO - 10.1016/S0021-9673(01)95796-6

M3 - Article

C2 - 1652592

AN - SCOPUS:0025849707

VL - 543

SP - 451

EP - 461

JO - Journal of Chromatography A

JF - Journal of Chromatography A

SN - 0021-9673

IS - C

ER -