Figure 5. Plot of enzyme rates in presence of high-affinity soy trypsin inhibitor. Source: Data from Ref. 14.

TNBS (trinitrobenzenesulfonic acid), the e-amino groups of lysine give a high blank value that may be removed by making the succinyl (19) or TV,TV-dimethyl (20) derivative of the protein. The latter is preferable for trypsinlike proteases.

Small synthetic molecules are also useful for assaying proteases. These give a change in spectrophotometric ab-sorbance as they are hydrolyzed, so a continuous assay with its advantages is possible. Table 1 lists a number of small molecule substrates. Most of these are applicable to serine and/or sulfhydryl proteases, with two exceptions: FAGLA is a substrate for metalloprotease (neutral protease), and Z-Gly-Phe is a carboxypeptidase substrate. Acidic protease may be assayed using a chromogenic peptide. Aminopeptidase is usually assayed using an amino acid derivative such as L-leucine-/?-naphthylamide.

Protease Assays. Most assays with protein substrates involve incubation for a set length of time, stopping the reaction with TCA (trichloroacetic acid), and measuring the amount of soluble peptide. Buffered TCA (0.11 MTCA, 18 g/L; 0.22 M sodium acetate, 18 g/L; and 0.33 M acetic acid, 19.8 g/L) gives superior enzyme linearity compared

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