Fluorogenic substrates for chymotrypsin with new fluorescent markers.
Several substrates for chymotrypsin Glt-Phe-NHRx have been synthesized, where RxNH2 are the compounds 3-aminocoumarin (2), 6-aminocoumarin (3), 3-acetamido-6-aminocoumarin (4), 3-acetamido-8-aminocoumarin (5), 7-amino-4-methyl-2-quinolinone (AMQ), (6). The fluorescence properties of the new substrates and those of the corresponding free amines were examined. The compound 7-glutarylphenylalaninamido-4-methyl-2-quinolinone (Glt-Phe-AMQ), (15), provided a new suitable substrate for chymotrypsin determination. The enzymatic release of the fluorophore AMQ was measured at lambda ex = 360 nm and lambda em = 435 nm. The Km of 15 was 0.5 mM and its kcat/Km ratio was 47 M-1 s-1. By using this substrate, the detection limit of chymotrypsin was 10 ng/ml.
D002918:Chymotrypsin / Q000378:metabolism*; D005456:Fluorescent Dyes; D006868:Hydrolysis; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D002918:Chymotrypsin