Dlpeptldase Horiuchl et al 1982

In a study of dipeptidase by Horiuchi et al. (1982), the tripeptide hippurylhisti-dylleucine (Hip-His-Leu) was used as a substrate, and the assay involved measuring the amount of hippuric acid released by the enzyme.

The substrate was separated from the product by reversed-phase HPLC (Nucleosil 7 C]8) using a mobile phase of methanol-10 mM KH2P04 (1:1) adjusted to pH 3.0 with phosphoric acid. Detection was at 228 nm. The separation of the components of the reaction mixture is shown in Figure 9.31.

The reaction mixture contained a phosphate buffer at pH 8.3 with NaCl and the substrate Hip-His-Leu. The reaction was incubated with the dipeptidase preparation for 30 minutes at 37°C and terminated with 3% metaphosphoric acid. The mixture was centrifuged, and a sample of the supernatant solution was injected onto the HPLC column for analysis. The results of an assay are shown in Figure 9.31. The appearance of the hippuric acid is taken as evidence of enzymatic activity. The rate of product formation is shown in Figure 9.32.

Peptidase preparations obtained from rat blood, lung, and kidney were chopped and homogenized, and clarified by centrifugation at 20,000g for 20 minutes. The supernatant solution served as the source of peptidase activity.

Figure 9.30 HPLC traces of solutions of Hip-Lys containing 75% human plasma incubated for 15 minutes. The substrate concentration was 1.02 mM X and Y represent unknown substances from plasma. (From Marceau et al., 1983.)

Figure 9.30 HPLC traces of solutions of Hip-Lys containing 75% human plasma incubated for 15 minutes. The substrate concentration was 1.02 mM X and Y represent unknown substances from plasma. (From Marceau et al., 1983.)

Was this article helpful?

0 0

Post a comment