Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phasse 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 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.