A NOVEL BIOLUMINOGENIC ASSAY FOR ALPHA-CHYMOTRYPSIN

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.