Preparation and Characterization of Guanidinated Trypsinogen and ∈-Guanidinated Trypsin

Reaction of bovine trypsinogen with 1-guanyl-3,5-dimethylpyrazole converted all 15 lysine residues to homoarginyl residues. Trypsin could not activate guanidinated trypsinogen but an acid protease from Aspergillus oryzae generated active ∈-guanidinated trypsin by cleaving the Har₆–Ile₇ bond in the modified zymogen and releasing the activation peptide Val-(Asp)₄-Har. After purification by affinity chromatography, the ∈-guanidinated enzyme contained 0.94–0.98 active site/molecule and exhibited the same specific activity toward benzoyl-L-arginine ethyl ester as β-trypsin.