Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phase high-performance liquid chromatography.
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 capillaries 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 endoproteinase Arg C preparations exhibited peptidase activity at Lys-15-Lys16 and at Lys16-Arg17 in addition to the expected cleavage at Arg-X bonds. ACTH peptide bond cleavage rates for Arg8-Trp9, Arg17-Arg-18, Lys15-Lys16, and Lys16-Arg17 were 1.46, 0.096, 0.57, and 0.029 mumol 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.
Krueger
R J
RJ
Department of Biochemistry and School of Biological Sciences, University of Nebraska, Lincoln 68583-0718.
Hobbs
T R
TR
Mihal
K A
KA
Tehrani
J
J
Zeece
M G
MG
D000324:Adrenocorticotropic Hormone / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D010450:Endopeptidases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D012697:Serine Endopeptidases; D013552:Swine
D010455:Peptides; D000324:Adrenocorticotropic Hormone; D010450:Endopeptidases; C044499:submandibular proteinase A; D012697:Serine Endopeptidases