The correction of reaction rates in continuous fluorometric assays of enzymes |
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| Institution: | 1. Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, T2N 1N4, Canada;2. Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA;1. The Institute for Drug Research, School of Pharmacy, Faculty of Medicine, Hebrew University, Jerusalem, Israel;2. Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel;3. Department of Oral Biology, The Goldschleger School of Dental Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel;4. Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia;5. Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia;6. Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova cesta 39, SI-1000 Ljubljana, Slovenia;7. Department of Materials Science and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv, Israel;1. Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, Devon EX1 2LU, UK;2. Royal Devon and Exeter NHS Foundation Trust, Exeter, Devon EX2 7JU, UK |
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| Abstract: | The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D–Arg-NH2, was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at μM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10− 4 × s− 1 were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH2, were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was − 2.96 × 10− 4 a.u μM− 1 × s− 1 that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor substrate] ÷ (final − initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of Km and overestimation of Vmax. |
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