Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane-Tricine buffer system.
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.
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Institute for Health Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan.
Katunuma
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D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives*; D011480:Protease Inhibitors / Q000032:analysis / Q000302:isolation & purification*; D011506:Proteins / Q000378:metabolism; D013194:Staining and Labeling; D014325:Tromethamine
D002021:Buffers; D011480:Protease Inhibitors; D011506:Proteins; D014325:Tromethamine; D010450:Endopeptidases; D005998:Glycine; C100184:tricine