Labeled proteinase inhibitors: versatile tools for the characterization of serine proteinases in solid-phase assays. |
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Authors: | C Breton-Maintier R Mayer D Richard-Molard |
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Affiliation: | Laboratoire de Microbiologie et Technologie Céréalières, INRA, Nantes, France. |
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Abstract: | Peptidyl chloromethyl ketones were used for the specific labeling of proteinases by attaching a biotin group to the N-terminal end of the peptide. Such labeled peptide inhibitors allowed the detection and quantitation of proteolytic enzymes immobilized on the plastic surface of a microtiter plate, as well as on nitrocellulose. The validity of these solid-phase assays was demonstrated using subtilisin Carlsberg as a model enzyme and biotinyl-epsilon-aminocaproyl-L-alanyl-L-alanyl-L-propyl-L-phenylal++ + anyl- chloromethyl ketone as a specific reagent. In addition to being usable for the screening of a particular proteinase in a large number of samples, these assays can be adapted for the analysis of specific proteolytic enzyme present in complex mixtures. |
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