The use of 6-(N-acetyl-L-phenylalanyl)-aminoluciferin as a novel subst
rate for alpha-chymotrypsin has been demonstrated. The kinetic paramet
ers determined are K-M = 0.38 mmol/L, k(cat) = 6.5 s(-1) and k(cat)/k(
M) = 17,100 (L/mols), The test principle of the coupled assay is the r
elease of aminoluciferin by enzymatic cleavage of 6-(N-acetyl-L-phenyl
alanyl)-aminoluciferin. Aminoluciferin is oxidized, with light emissio
n. 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 b
een synthesized as an example for a new class of highly sensitive subs
trates, By modification of the peptide residue these new substrates ma
y be suitable for ultrasensitive detection of different proteinases.