Detection of protease inhibitors using substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
A technique is described to detect the activity of protease inhibitors present in sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAG) containing a copolymerized enzyme substrate. The method involved (1) incorporation of substrate (gelatin or casein) into the SDS-PAG at the time of casting; (2) electrophoresis of the protease inhibitors in the presence of SDS; (3) removal of SDS by washing the gel in 2.5% (w/v) Triton X-100; (4) incubation of the gels in a solution containing the proteolytic enzyme at 37 degrees C for 16 h; and (5) staining undigested substrate with amido black. Standard inhibitors such as bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (SBTI), alpha 1-antitrypsin inhibitor, and a protease inhibitor derived from human articular cartilage have been examined by this method and displayed sharp inhibition bands when the gels were treated with bovine trypsin, chymotrypsin, or other enzymes. The technique cannot be used for precise quantification of protease inhibitors. However, there is a relationship between the concentration of inhibitor used and the intensity of staining. By this means, it was possible to estimate the smallest amount of inhibitor that could be detected (against a particular enzyme) under a given set of conditions. Inhibition was detected when 10 ng of SBTI or 20 ng of BPTI were applied to the gels; human alpha 1-protease inhibitor could be detected at a level of 2-3 micrograms. The technique was used to investigate the effectiveness of the human cartilage inhibitor against a variety of proteolytic enzymes, including thermolysin, Pronase, neutral protease, elastase, protease VII, pepsin, bacterial collagenase, protease IV, and papain.
D000818:Animals; D002358:Cartilage, Articular / Q000032:analysis; D002364:Caseins; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005780:Gelatin; D006801:Humans; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000302:isolation & purification*
D002364:Caseins; D011480:Protease Inhibitors; D005780:Gelatin; D010447:Peptide Hydrolases