A flow microcalorimetric method for enzyme activity measurements: Application to dihydrofolate reductase |
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| Institution: | 1. Research Unit for Functional Biomaterials, Department of Science and Environment, Roskilde University, 1 Universitetsvej, Build. 28.C, DK-4000 Roskilde, Denmark;2. Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark;1. Department of Science and Technology, Ministry of Science and Technology, Govt. of India, India;2. Amity institute of Biotechnology, Amity University Uttar Pardesh, Sector-125, Noida, U.P. 201313, India;3. CSIR-Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research, New Delhi, India;4. Academy of Scientific and Innovative Research (AcSIR), New Delhi, India;5. Amity institute of Molecular Medicine & Stem Cell Research, Amity University Uttar Pardesh, Sector-125, Noida, U.P., 201313, India;1. Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10 000 Zagreb, Croatia;2. Glycobiology Department, Genos Ltd., Hondlova 2, 10 000 Zagreb, Croatia |
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| Abstract: | A flow microcalorimetric method was developed for the analysis of enzymatic activities in crude tissue homogenates. It can be applied whenever a heat exchange is involved in an enzymatic reaction. The consequent sensitivity obviously depends on the enthalpy variation observed. Dihydrofolate reductase was chosen as an example; this enzyme is the molecular target of methotrexate, a widely used anticancer agent. This calorimetric method, whose sensitivity limit is 1.48 × 10−4 units of dihydrofolate reductase per milliliter of reactant medium, allows enzyme activity measurements in tissues with low dihydrofolate reductase levels. A few examples of measurements in animal tissues are given. These measurements are of some interest; indeed, increased activity and increased levels of this enzyme are two of the mechanisms which may explain resistance to methotrexate. |
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