A novel bioluminogenic assay for alpha-chymotrypsin

The use of 6-(N-acetyl-L-phenylalanyl)-aminoluciferin as a novel substrate for alpha-chymotrypsin has been demonstrated. The kinetic parameters determined are KM = 0.38 mmol/L, kcat = 6.5 s-1 and kcat/kM = 17,100 (L/mol s). The test principle of the coupled assay is the release of aminoluciferin by enzymatic cleavage of 6-(N-acetyl-L-phenylalanyl)-aminoluciferin. Aminoluciferin is oxidized, with light emission, by firefly luciferase (Photinus pyralis) and can be quantified in a luminometric assay. The detection limit for chymotrypsin was found to be 0.3 ng per assay. 6-(N-acetyl-L-phenylalanyl)-aminoluciferin has been synthesized as an example for a new class of highly sensitive substrates. By modification of the peptide residue these new substrates may be suitable for ultrasensitive detection of different proteinases.