A simple, combined fluorogenic and chromogenic method for the assay of proteases in gingival crevicular fluid

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Abstract
Substrate impregnated paper discs were prepared using peptidyl derivatives of 7‐amino‐4‐trifluoromethylcoumarin (AFC). After incubation with test solutions, the green, UV‐induced fluorescence of AFC liberated by enzyme activity was distinguishable from the blue‐violet fluorescence of the substrates. The AFC could then be coupled with p‐dimethylaminocinnamaldehyde to form a colored Schiff base. Semi‐quantitative assessments of disc fluorescence and color were made by comparison with AFC/substrate standards. Assays with discs impregnated with MeOSuc‐Ala‐Ala‐Pro‐Val‐AFC, Z‐Gly‐Gly‐Arg‐AFC and Ala‐Pro‐AFC for elastase‐, trypsin‐, and dipeptidyl peptidase (DPP) IV‐like activities respectively were evaluated using purified DPP IV and 100 eluates of crevicular fluid collected on filter paper strips from 10 gingivitis and periodontitis patients. The results showed that, within their working ranges, scores of disc fluorescence and color were reasonably accurate and reliable by comparison with enzyme activities measured in parallel quantitative fluorimetric assays with the same substrates. Using disc color, which was more sensitive than fluorescence, it was generally possible to measure all three enzyme activities in crevicular fluid samples from 5 periodontitis patients with varying degrees of gingival inflammation and pocketing. Disc color assays require no special apparatus and could be used for enzyme estimations in the clinical setting.