title: Automated enzyme assays by use of a centrifugal analyzer with fluorescence detection.
abstract: The first commercially available centrifugal analyzer having fluorescence detection capability was used to develop kinetic fluorometric assays for several proteinases. The substrates were all synthetic oligopeptides incorporating 7-amino-4-trifluoromethylcoumarin, a molecule that can be detected either spectrophotometrically or fluorometrically. We thus compared the centrifugal analyzer spectrophotometric and fluorometric detection systems, finding fluorescence detection to be 50-fold more sensitive. We also compared the sensitivity of the fluorescence detector to that of a conventional spectrofluorometer by determining the minimum detection limit for each enzyme on both instruments; we found them to be similar in sensitivity. As an illustrative application, we measured the cathepsin B-like activity in serum samples from 55 women. The median enzyme activity of women taking oral contraceptives and pregnant women was increased two- and threefold, respectively, over the control group (about 5% CV within run, and 10% CV between runs).
mesh_terms: D001322:Autoanalysis; D002401:Cathepsin B; D002403:Cathepsins / Q000097:blood; D002498:Centrifugation / Q000379:methods; D003276:Contraceptives, Oral; D005260:Female; D006801:Humans; D010447:Peptide Hydrolases / Q000032:analysis*; D011247:Pregnancy; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods
chemical_list: D003276:Contraceptives, Oral; D002403:Cathepsins; D010447:Peptide Hydrolases; D002401:Cathepsin B