A simple method for selection of trypsin chromogenic substrates using combinatorial chemistry approach
Section snippets
Materials and methods
Peptide synthesis. Peptides for kinetic studies and peptide library were synthesized manually by the solid-phase method using Fmoc chemistry, as described previously [8]. TentaGel S RAM (substitution 0.23 meq/g) (RAPP Polymere, Germany) was used as a support. The amino acid derivatives used for the synthesis were as follows: Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Pro, Fmoc-Val, Fmoc-Arg(Pbf), Fmoc-Lys(Boc), Fmoc-Ser(tBu), Fmoc-Asp(OtBu), Fmoc-Glu(OtBu), and Fmoc-Tyr(tBu). 5-Amino-2-nitrobenzoic acid
Results and discussion
The deconvolution of the peptide library (consisting of 114=14641 peptides) against bovine β-trypsin is summarized in Fig. 1. The results obtained indicate that the aromatic Phe residue present in position P4 of the synthesized peptide library displays the strongest interaction with the enzyme investigated. Interestingly, the other aromatic residue (Tyr) present in the library had a significantly lower impact on the substrate activity. With the Phe residue fixed in position P4, sublibraries
Acknowledgements
This work was supported by the Polish State Committee for Scientific Research (KBN), Grant No. 1007/T09/2003/24, and by the University of Gdańsk (Grant No. BW-8000-5-0324-3).
References (12)
Design, Chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III)
Biochem. Biophys. Res. Commun.
(2000)Chromogenic substrates of bovine–trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme
Biochem. Biophys. Res. Commun.
(2001)Synthesis and physical characterization of a P1 arginine combinatorial library, and its application to the determination of the substrate specificity of serine peptidases
Bioorg. Med. Chem. Lett.
(2002)Selection of low-molecular mass trypsin and chymotrypsin inhibitors based on the binding loop of CMTI-III using combinatorial chemistry approach
Biochem. Biophys. Res. Commun.
(2003)Generation and use of nonsupport-bound peptide and peptidomimetic combinatorial libraries
Methods Enzymol.
(1996)The preparation and properties of two new chromogenic substrates of trypsin
Arch. Biochem. Biophys.
(1961)
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2008, Analytical BiochemistryCitation Excerpt :The peptide library and individual peptides for kinetic studies were synthesized manually by the solid-phase method using fluorenyl-9-methoxycarbonyl (Fmoc) chemistry as described previously [12].