A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is
reported. Inhibitor samples were separated on a protein substrate-SDS-polyacrylamide gel in a Tris-Tricine
buffer system that improves the separation and identification of peptides and low-molecular-weight proteins.
After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein
substrate. The inhibitor bands, which were protected from proteolysis by the target proteases, were stained.
Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases
inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for
tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands
in the white background when the gels were treated with the target proteases. Some significant applications
of this method are introduced. This method is an ideal system for discovering new protease inhibitors in
small natural samples. ?? 2003 Elsevier Inc. All rights reserved.
