Abstract
Methods for the demonstration of peptidase activities “in situ” using natural peptides as substrates are described and evaluated. They are based on the use of the l-amino acid oxidase of Vipera ammodytes venom (or of similar snake venom) which is able to catalyze efficiently the oxidation of Leu, Met, Phe, Ileu, Tyr, and Try set free by the action of the particular peptidase(s). Accordingly, peptides from which the mentioned aminoacids are set free can be used as substrates. Cryostat sections adherent to slides are employed which can be preextracted with chloroform-acetone mixture (āā, 4°C, 5 min) to eliminate lipids. All substances needed for this multistep reaction are prepared and applied in a suitable gel layer which impedes the diffusion of soluble peptidases and also of l-amino acids set free by their action. The enzyme activity is assessed from the difference between the coloration of a pair of parallel sections one of which was incubated with the substrate and the other without it.
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