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Fluorogenic substrates for chymotrypsin with new fluorescent markers.

G Kokotos, C Tzougraki

    International Journal of Peptide and Protein Research
    |August 1, 1986
    PubMed
    Summary

    New fluorescent substrates were developed for quantifying chymotrypsin, an important enzyme. The substrate 7-glutarylphenylalaninamido-4-methyl-2-quinolinone (Glt-Phe-AMQ) allows sensitive chymotrypsin detection down to 10 ng/ml.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Fluorescence Spectroscopy

    Background:

    • Chymotrypsin is a key serine protease involved in various physiological and pathological processes.
    • Sensitive and specific assays are crucial for chymotrypsin activity determination.
    • Fluorescent substrates offer advantages in sensitivity and real-time monitoring of enzymatic activity.

    Purpose of the Study:

    • To synthesize and characterize novel fluorescent substrates for chymotrypsin.
    • To evaluate the suitability of these substrates for enzyme activity assays.
    • To establish a sensitive method for chymotrypsin detection using a developed substrate.

    Main Methods:

    • Synthesis of various coumarin and quinolinone-based substrates.
    • Characterization of fluorescence properties of synthesized compounds.
    • Enzymatic assays using chymotrypsin and the developed substrates.
    • Determination of kinetic parameters (Km, kcat/Km) and detection limits.

    Main Results:

    • Several novel substrates were synthesized, including derivatives of aminocoumarin and 7-amino-4-methyl-2-quinolinone (AMQ).
    • The substrate 7-glutarylphenylalaninamido-4-methyl-2-quinolinone (Glt-Phe-AMQ) demonstrated suitability for chymotrypsin determination.
    • Enzymatic release of AMQ was measured (lambda ex = 360 nm, lambda em = 435 nm).
    • Glt-Phe-AMQ exhibited a Km of 0.5 mM and a kcat/Km of 47 M-1 s-1.
    • A detection limit of 10 ng/ml for chymotrypsin was achieved using Glt-Phe-AMQ.

    Conclusions:

    • 7-glutarylphenylalaninamido-4-methyl-2-quinolinone (Glt-Phe-AMQ) is a highly effective and sensitive substrate for chymotrypsin.
    • The developed assay allows for precise quantification of chymotrypsin activity.
    • This substrate provides a valuable tool for biochemical research and diagnostics involving chymotrypsin.

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