Detection of cathepsin B‐ and L‐, elstase‐, tryptase‐, trypsin‐, and dipeptidyl peptidase IV‐like activities in crevicular fluid from gingivitis and periodontitis patients with peptidyl derivatives of 7‐amino‐4‐trifluoromethyl coumarin

Abstract

Crevicular fluid samples were collected from 20 gingivitis and periodontitis patients using filter paper strips; these were then eluted into buffer. Portions of each sample were combined and the activities of this pooled eluate against different peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin (AFC) were examined with respect to their pH profiles and effector responses. Cathepsin B- and L-like activity was detected with Bz-Val-Lys-Lys-Arg-AFC; elastase-like activity with MeOSuc-Ala-Ala-Pro-Val-AFC; tryptase-like activity with Z-Ala-Ala-Lys-AFC; trypsin-like activity with Z-Gly-Gly-Arg-AFC; and dipeptidyl peptidase (DPP) IV-like activity with Ala-Pro-AFC. The selectivity and sensitivity of these assays were improved by choice of appropriate conditions. The cathepsin B- and L-, elastase-, tryptase-, and trypsin-like activities all had properties consistent with those from host sources, whilst partial inactivation of the DPP IV-like activity by heat treatment (60.degree. C for 30 min) suggested that it may have represented a mixture of human and Bacteroides gingivalis enzymes. Individual patient eluates showed wide variations in enzyme concentrations, but generally elastase-like activity was by far the highest. The sensitivity of the assays with AFC-linked substrates was such that it should prove possible to measure all five different types of activity in crevicular fluid samples from local periodontal disease sites.