Affinity Chromatography of Proteolytic Enzymes

  • V. M. Stepanov


Two types of specific ligands attached to Sepharose or macroporous silica were used for affinity chromatography of proteinases: binding site and catalytic site directed. Several binding site-directed ligands were used. Peptides, such as 6-aminohexanoyl(6-Ahx)-D-PheOCH3 or 6Ahx-L-Phe-D-Phe-OCH3 and their analogs, were used for purification of swine, bovine, or horse pepsins, calf chymosin, and aspergillopepsin A (carboxylic proteinase from Abp. awamoti) (1,2). Pronounced electrostatic effects were detected for the sorbents containingthese ligands(Ki was about 1-3 mM) (3). Peptide antibiotics, such as Gramicidin S (cyclopeptide containing hydrophobic amino acids), might be regarded as a substrate analog. Due to the specific conformation and the presence of D-amino acid, it was stable to proteolysis and acted as a weak inhibitor for various proteinases (Ki of the order 1-5 mM). Other bacterial polypeptides might also be used as the ligands. Various pepsins (swine, bovine, horse) and aspergillopepsins A and F (from Abp. boetidus) were purified on these sorbents (4,5), as well as subtilisin, metalloproteinase (6), and intracellular serine proteinase from Bac. bubtitis (7). Gramicidin S-Sepharose also was used to purify carboxylic proteinase from the carnivorous plant Nepenthes Also, mono-2,4-dinitrophenyl-hexamethylenediamine and its analogs might serve as the ligands for hydrophobic chromatography of carboxylic proteinases (8).


Affinity Chromatography Catalytic Site Hydrophobic Amino Acid Specific Ligand Weak Inhibitor 
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Copyright information

© Plenum Press, New York 1978

Authors and Affiliations

  • V. M. Stepanov
    • 1
  1. 1.Institute of Genetics and Selection of Industrial MicroorganismsMoscowUSSR

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