Automated enzyme assays by use of a centrifugal analyzer with fluorescence detection.

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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).