Sensitive method to identify and characterize proteinases in situ after SDS-PAGE

Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and dial cult tasks. Especially di fficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based f luorogenic substrates. To rest the sensitivity of the technique, we used tr ypsin as our model system. Filter paper impregnated with rhodamine-based fl uorogenic substrates was placed on a gel, and bands of fluorescence origina ting from specific proteinases,were visualized in real lime. The method is very sensitive; picogram amounts of trypsin can be detected. The method sho uld be very general, in that even proteinases whose substrates require amin o acids C-terminal to the cleavage site may be identified and characterized . The results allow one to obtain not only information on the substrate spe cificity of a specific enzyme but also information about its molecular weig ht.