Characterizing and optimizing protease/peptide inhibitor interactions, a new application for spot synthesis

A new method is presented that uses parallel peptide array synthesis on cel lulose membranes to characterize protease/peptide inhibitor interactions. A peptide comprising P5-P4' of the third domain of turkey ovomucoid inhibito r was investigated for both binding to and inhibition of porcine pancreatic elastase. Binding was studied directly on the cellulose membrane, while in hibition was measured by an assay in microtiter plates with punched out pep tide spots. The importance of each residue for binding or inhibition was de termined by substitutional analyses, exchanging every original amino acid w ith all other 19 coded amino acids. Seven hundred eighty individual peptide s were investigated for binding behavior to porcine pancreatic elastase, an d 320 individual peptides were measured in inhibition experiments, The resu lts provide new insights into the interaction between the ovomucoid derived peptide and subsites in the active site of elastase, Combining these data with length analysis we designed new peptides in a stepwise fashion which i n the end not only inhibited elastase 400 times more strongly than the orig inal peptide, but are highly specific for the enzyme. In addition, the opti mized inhibitor peptide was protected against exopeptidase attack by substi tuting D-amino acids at both termini.