Purification of pronase by ion-exchange chromatography gave four proteolytically active fractions. Fraction A2 contained an endopeptidase that attacks poly l-valine. Fraction B contained an endopeptidase, an aminopeptidase and carboxypeptidases. The activities against hippuryl-L-arginine and hippuryl-L-phenylalanine could be inhibited to a considerable extent by di-isopropyl phosphorofluoridate and by EDTA. Fraction C contained an endopeptidase resembling bovine trypsin. The pure enzyme was completely inactivated by di-isopropyl phosphorofluoridate and pancreatic trypsin inhibitor and to about 90% by other naturally occurring trypsin inhibitors. Fraction D contained an apparently homogeneous endopeptidase, inhibited by diisopropyl phosphorofluoridate, that adsorbed to and hydrolysed elastin. The activity of all these fractions was tested qualitatively against a wide range of small peptides and synthetic substrates.
