Fluorometric assay for pancreatic cholesterylester hydrolase |
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| Authors: | A Joutti L Kotama J A Virtanen P K Kinnunen |
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| Affiliation: | 1. REQUIMTE/LAQV, ISEP, Polytechnic of Porto, Rua Dr. António Bernardino de Almeida 431, 4249-015, Porto, Portugal;2. Nutrition & Metabolism, CINTESIS@RISE, NOVA Medical School, Faculdade de Ciências Médicas, NMS, FCM, Universidade NOVA de Lisboa, Campo dos Mártires da Pátria 130, 1169-056, Lisboa, Portugal;3. Center for Research in Health Technologies and Information Systems, Rua Dr. Plácido da Costa, 4200-450, Porto, Portugal;4. Faculty of Medicine, University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319, Porto, Portugal;2. Institute for Clinical Chemistry and Clinical Pharmacology, University Clinics Bonn, D-53127 Bonn, Germany;1. Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, DK-5230, Odense M, Denmark;2. Department of Molecular Biology and Genetics, University of Aarhus, DK-8000, Aarhus C, Denmark;3. Department X-Ray Microscopy, Helmholtz-Zentrum Berlin, Albert-Einstein-Str. 15, 12489, Berlin, Germany;4. Niels Bohr Institute, University of Copenhagen, Universitetsparken 5, 2100, Copenhagen, Denmark;1. Department of Chemistry, Jahangirnagar University, Savar, Dhaka, 1342, Bangladesh;2. Pharmaceutical Sciences Research Division, BCSIR Laboratories, Dhaka, Dhaka, 1205, Bangladesh;3. Biological Research Division, BCSIR Laboratories, Dhaka, Dhaka, 1205, Bangladesh;4. Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka, 1000, Bangladesh;1. College of Food Science and Technology, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Products Processing and Safety, Guangdong Provincial Engineering Technology Research Center of Marine Food, Guangdong Province Engineering Laboratory for Marine Biological Products, Zhanjiang 524088, China;2. Key Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China;3. Co-Innovation Center of Jiangsu Marine Bio-Industry Technology, Jiangsu Ocean University, Lianyungang 222005, China;4. Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Dalian 116034, China |
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| Abstract: | A fluorescent cholesterylester analogue, cholesteryl 6-pyrenylhexanoate (ChPH), was used as a substrate for pancreatic cholesterylester hydrolase (CEH, EC 3.1.1.13). The substrate consisted of ChPH in egg phosphatidylcholine stabilized microemulsion with the aqueous phase containing deoxycholate below its critical micellar concentration. Due to the high local concentration of the pyrene moiety in the ChPH phase the fluorescence emission due to monomeric pyrene (IM) is greatly exceeded by the excimer fluorescence intensity (IE). Upon reacting with CEH 6-pyrenylhexanoic acid and free cholesterol are formed. The fluorescent product, 6-pyrenylhexanoic acid, is transferred into the aqueous phase containing deoxycholate, thus resulting in an enhanced fluorescence due to monomeric pyrene. CEH activity can thus be assessed directly by monitoring IM vs. time without product separation. Useful assay conditions were found to be 10 microM ChPH, 0.1 microM egg phosphatidylcholine, 2 mM sodium deoxycholate at 25 degrees C and pH 6.5-7.0. |
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