A molecular probe with l-phenylalanine p-nitroanilide and l-lysin 4-methylcoumaryl-7-amide, in which these amino acid derivatives are connected through a succinic-acid spacer, was prepared. Trypsin and papain were detected by blue-fluorescence emission of generated 7-amino-4-methylcoumarin (AMC). α-Chymotrypsin and nattokinase were detected from both the blue-fluorescence emission of AMC and the UV absorbance of p-nitroaniline. In addition, different time courses of p-nitroaniline and AMC were observed between the reaction of P1 with α-chymotrypsin and that with nattokinase. In the case of nattokinase, both the fluorescence emission and UV absorbance slowly increased. In contrast, the increasing UV absorbance was saturated at the early stage of the reaction of the present probe with chymotrypsin, whereas the fluorescence emission continuously increased in the following stages.
Keywords: analysis; chromogenic molecular probe; molecular fluorescence probe; protease.