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.
Department of Dermatology and Andrology, Justus Liebig University, Giessen, Germany.
T
T
Monsees
R
R
Geiger
W
W
Miska
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