We hypothesize that degradation of hemoglobin and other host plasma proteins in the gut of schistosome parasites is carried out by a cascade or network of proteolytic enzymes. To map the biochemical steps involved in this pathway, we are initially focusing on two proteases: schistosome legumain (asparaginyl endopeptidase) and schistosome cathepsin B. These two proteases are thought to represent an initial zymogen activation and subsequent host protein degradation segment of the cascade. To circumvent difficulties in evaluating schistosome metabolic pathways, we are utilizing several new synthetic and biochemical techniques to dissect biologic function by "chemical knockout". The substrate specificity of both enzymes will first be mapped in detail utilizing a combination of diverse combinatorial fluorogenic peptide substrate libraries and substrate phage display. Substrate preference at the amino acid positions P3-P3' will give clues to potential protein substrate cleavage sites that can be validated by direct analysis of protein degradation products. Using 2D gel electrophoresis and mass spectrometry, major natural substrates of cathepsin B will be confirmed by analyzing regurgitant proteins from adult worm guts for protein profile changes in the presence or absence of cathepsin B or legumain-specific inhibitors. Preliminary studies have confirmed the feasibility of this approach, and identified several putative plasma protein substrates were identified. Cleavage patterns of these substrates can now be compared to the predicted substrate specificity of the proteases being evaluated. To ensure we can carry out chemical knockout with appropriate specificity, diverse combinatorial inhibitor libraries have been produced for both enzymes, and will next be screened for protease- specific inhibitors that can be used to confirm expected, or uncover unexpected biological functions. Recently developed radiolabeled or fluorescently "tagged" inhibitors will also be used to identify, localize, and quantitate both enzymes in schistosome extracts, gut regurgitant material, and gut epithelium. The results of these studies should have import not only in unraveling the pathway of schistosome blood protein digestions, but providing a technical approach to aid investigators studying the similar cascades recently identified in Fasciola and Haemonchus and likely to be present in other blood feeding nematode and trematode parasites.