FRETS Peptide Libraries

Each substrate (Code 3701-v – Code 3719-v) in the FRETS-25Xaa series contains a highly fluorescent 2-(N-methylamino)benzoyl (Nma) group linked to the side chain of the amino-terminal D-2,3-diamino propionic acid (D-A2pr) residue, which is efficiently quenched by a 2,4-dinitrophenyl (Dnp) group linked to the function of Lys. Xaa represents a fixed position of each of the 19 natural amino acids excluding Cys (noted in product name Code 3701-v – Code 3719-v). A mixture of 5 amino acid residues (P, Y, K, I, and D) is at the Yaa position along with a mixture of 5 amino acid residues (F, A, V, E, and R) at the Zaa position for each fixed Xaa. This provides a peptide mixture of 25 combinations of each Xaa series resulting in a combinatorial library totaling 475 peptide substrates. Both Nma and Dnp groups are linked to the side chain of the individual residues, allowing for the determination of the cleavage site by a specific enzyme through mass spectrometric analysis and Edman degradation as well. The enzymatic reaction should be terminated by using a proper inhibitor ( Leupeptin , E-64 or Pepstatin A ).

We have confirmed that FRETS-25Xaa series are effectively used for the assay of numerous proteases such as trypsin, chymotyrpsin, elastase, thrombin, papain, calpain, pepsin and thermolysin. However, they did not work well for the assay of caspase-3, probably because they have only three changeable sites (Zaa-Yaa-Xaa) in each substrate (deficiency of P4 site). This fact implies that FRETS-25Xaa might not be applicable to the assay of an enzyme with wide range interacting sites with substrate.

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