PORTION-MIXING PEPTIDE LIBRARIES OF QUENCHED FLUOROGENIC SUBSTRATES FOR COMPLETE SUBSITE MAPPING OF ENDOPROTEASE SPECIFICITY

A solid-phase assay for the complete subsite mapping of the active sit e of endoproteases has been developed. A library of resin-bound protea se substrates was synthesized both on kieselguhr-supported polyamide r esin and on a polyethylene glycol-poly-(N,N-dimethylacrylamide) copoly mer type of resin that allows proteases to diffuse into the interior a nd perform their catalytic activity. Anthranilic acid and 3-nitrotyros ine were used as an efficient donor-acceptor pair for the resonance en ergy transfer. The synthesis was performed in a manual library generat or that allows simple wet mixing of the beads and parallel washing pro cedures. After treatment with subtilisin Carlsberg, fluorescing beads were collected and subjected to peptide sequencing, affording the pref erred sequences, their cleavage bond, and a semiquantitative estimatio n of the turnover. A statistical distribution of preferred amino acids was obtained for each subsite. The result was compared with data from kinetic studies in solution.