A novel bioluminogenic assay for α-chymotrypsin

✍ Scribed by Monsees, T. ;Geiger, R. ;Miska, W.

• Publisher: John Wiley and Sons
• Year: 1995
• Weight: 383 KB
• Volume: 10
• Category: Article
• ISSN: 0884-3996
• DOI: 10.1002/bio.1170100404

✦ Synopsis

The use o f 6-(N-acetyl-~-phenylalanyl)-aminoluciferin as a novel substrate for achymotrypsin has been demonstrated. The kinetic parameters determined are K M = 0.38 mmol/L, kcat = 6.5 s-' and kcat/kM = 17,100 (L/mol s). The test principle o f the coupled assay is the release o f aminoluciferin by enzymatic cleavage o f 6 -( Nacetyl-L-phenylalany1)-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 t o be 0.3ng per assay. 6-(Nacetyl-L-phenylalany1)-aminoluciferin has been synthesized as an example for a new class o f highly sensitive substrates. B y modification o f the peptide residue these new substrates may be suitable for ultrasensitive detection o f different proteinases.