Plasma carboxypeptidase N degrades and therefore inactivates bradykinin. This activity may have a role in the regulation of inflammatory peptides. The HPLC method developed for its assay uses the dipeptide hippuryllysine (Hip-Lys) as the substrate and measures activity by measuring the release of hip-puric acid.
The separation of substrate from the product was carried out by reversed-phase HPLC (CJ8 /xBondapak) using a mobile phase of a 1:4 mixture of methanol and 0.001 M K2HP04 and H3PO4 (pH 3.0). The column was protected by a precolumn packed with Corasil. The column was eluted isocrati-cally, and detection was at 230 nm. The separations obtained under these conditions are shown in Figure 9.29.
The reaction mixture contained Hepes [N-(2-hydroxyethyl)piperazine-N1-2-ethanesulfonic acid] buffer (pH 7.75) with NaCl and the substrate Hip-Lys in a volume of 500 fxL. The plasma was added to start the reaction, and the
Hip-Lys Hippuric Acid
5 10min 0
Figure 9.29 HPLC traces of standard solutions (32 /xg/mL) of Hip-Lys (HL) and hippuric acid (HA). (From Marceau et a)., 1983.)
reaction was terminated by the addition of absolute ethanol. The mixture was clarified by centrifugation, and the supernatant solution applied to the HPLC for analysis. The result of a 15-minute incubation is shown in Figure 9.30.
The carboxypeptidase was prepared from plasma.
Was this article helpful?