Multiple Column Synthesis of Quenched Solid-Phase Bound Fluorogenic Substrates for Characterization of Endoprotease Specificity

Abstract

A method for multiple column peptide synthesis of resin-bound fluorogenic protease substrates, which are subsequently used in a solid-phase assay for the complete subsite mapping of the active site of endoproteases, is described. Substrate libraries containing anthranilic acid and 3-nitrotyrosine as an efficient donor-acceptor pair for long-range resonance energy transfer were synthesized on kieselguhr-supported polyamide resin and on PEG-polyamide resin, both permitting proteases to diffuse into the interior. The synthesis was performed in a manual library generator that allows simple wet-mixing of the beads and parallel washing procedures. The library was treated with the proteolytic enzyme subtilisin Carlsberg, and fluorescent beads on a background of dark beads were collected manually. The preferred sequences, their scissile bonds, and semi-quantitative estimations of their turnover were determined by sequence analysis of the resin-bound peptides. For each subsite, a statistical distribution of preferred amino acids was obtained. Amino acid sequences that could not be hydrolyzed by extensive treatment with subtilisin Carlsberg were also identified.