Abstract 1276 Development of Methods to Test and Characterize Proteases in Human Saliva

Proteases play critically important roles in humans and abnormal activity is linked to a number of diseases. The proteases in saliva play a role in digestion of food and protection; abnormal activity can lead to periodontal disease among others. These enzymes are difficult to assay. The goal of this project was to characterize the activity of proteases by a variety of methods: Thin Layer Chromatography (TLC), SDS PAGE, and zymography. Methods were vetted by using pure proteases and then adapted for testing for protease activity in saliva. Several pure proteins were mixed with the three major proteases of digestion (chymotrypsin, trypsin or pepsin) over time: portions were removed and spotted on TLC plates or run on SDS PAGE. It was observed that new spots appeared with higher Rf values on TLC plates when proteases were added, and these spots were not present if the protease was heated prior to use, or a specific inhibitor was added. For SDS PAGE, new and smaller molecular weight (MW) bands appeared when pure proteins were mixed with the proteases which demonstrated protease activity. Both methods can demonstrate protease activity. These methods, however, yield limited information if there is a mixture of proteases as expected in saliva. For this reason, zymography was used. In this method, a protein is embedded in an SDS PAGE gel and proteases are run on the gel which is then washed and incubated and finally stained with Coomassie blue which yields a blue color with virtually all proteins. Proteases migrate on the gel to their respective molecular weights and when active, produce a clear spot as the embedded protein is degraded. The method was tested successfully with pure proteases and pure proteins and clear spots appeared at the expected MW of the protease. The method was also successful when a protein extract prepared from a natural source (ex. chicken extract) was embedded as the protein and a protease was added. When the method was repeated with a concentrated saliva extract and a pure protein embedded in the gel, there were proteases, and the MW was determined. This result suggests that while all three methods, TLC, SDS PAGE and zymography, can successfully detect protease activity, zymography yields the number and MW of proteases in a mixture of them. This method can be applied to characterizing the proteases in saliva on various food proteins.