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  • Title: Development of sensitive cathepsin G fluorogenic substrate using combinatorial chemistry methods.
    Author: Lesner A, Wysocka M, Guzow K, Wiczk W, Legowska A, Rolka K.
    Journal: Anal Biochem; 2008 Apr 15; 375(2):306-12. PubMed ID: 18261971.
    Abstract:
    In the current work, a synthesis of new sensitive fluorescence substrates of cathepsin G is reported. The substrate sequence was selected using combinatorial chemistry methods. The starting structure of chromogenic cathepsin G substrate Ac-Phe-Val-Thr-Gnf-ANB-NH(2), where Gnf stands for 4-guanidine-l-phenylalanine, was modified by replacing the acetyl moiety with a residue of 7-methoxycoumarin-4-yl acetic acid (Mca) that served as a fluorescence donor. An amide of amino benzoic acid (ANB-NH(2)) was used as an acceptor. This peptide, exhibiting effective fluorescence resonance energy transfer (FRET) phenomena, was used as a starting structure to construct the library Mca-Phe-Val-Thr-Gnf-X(1)-X(2)-ANB-NH(2), where in both variable X positions all proteinogenic amino acid residues except Cys were introduced. Deconvolution of such a library, performed by the iterative method in solution, revealed prime site preferences of cathepsin G. Finally, the most susceptible sequence, Mca-Phe-Val-Thr-Gnf-Ser-Trp-ANB-NH(2), was selected. The determined value of the specificity constant (k(cat)/K(M) = 252 x 10(3)M(-1)xs(-1)) was two orders of magnitude higher than that obtained for the parent compound. By the use of this substrate, we were able to detect as little as 70 pM of the enzyme studied.
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