A new approach to the use of fluorogenic dinitrophenyl-containing substrates for determining the proteolytic activity of aspartyl proteinases

A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.