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.
Monsees
T
T
Department of Dermatology and Andrology, Justus Liebig University, Giessen, Germany.
Geiger
R
R
Miska
W
W
D000818:Animals; D002918:Chymotrypsin / Q000032:analysis* / Q000378:metabolism; D049454:Firefly Luciferin / Q000031:analogs & derivatives / Q000378:metabolism; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D008156:Luciferases; D008163:Luminescent Measurements; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity
C091258:6-(N-acetylphenylalanyl)aminoluciferin; D007202:Indicators and Reagents; D010649:Phenylalanine; D049454:Firefly Luciferin; D008156:Luciferases; D002918:Chymotrypsin