Automated enzyme assays by use of a centrifugal analyzer with fluorescence detection.
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).
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
D003276:Contraceptives, Oral; D002403:Cathepsins; D010447:Peptide Hydrolases; D002401:Cathepsin B