A high pressure liquid chromatography-based assay for glutathione-S-transferase class distinction assay |
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| Institution: | 1. Global Discovery Chemistry/Oncology & Exploratory Chemistry, Novartis Institutes for Biomedical Research, Klybeckstrasse 141, CH-4057 Basel, Switzerland;2. Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA;3. Global Discovery Chemistry/Oncology & Exploratory Chemistry, Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA;1. School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, PR China;2. School of Pharmacy, China Medical University, Shenyang, Liaoning 110122, PR China;3. School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China;1. Department of Microbiology, College of Natural Sciences, Dankook University, Cheonan 31116, Republic of Korea;2. Department of Clinical Laboratory Science, Semyung University, Jecheon 27136, Republic of Korea;3. Department of Biological Sciences, Sookmyung Women''s University, Seoul, 04310, Republic of Korea |
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| Abstract: | In order to expedite the process of classification of the members of the family of glutathione-S-transferases (GSTs) high performance liquid chromatography with photodiode array detection (HPLC-PDA) was used as a means for measuring enzymatic activity. The GST chosen for the development of the HPLC-PDA technique was from equine liver (E-GST). The characterizing substrates, ethacrynic acid (EA) and bromosulfophthalein (BSP), along with previously gathered characterization data allowed for the distinction of α, μ or π-class enzymes. In an initial characterization of the previously unclassified E-GST it was determined that the enzyme was of the π-class with specific activities of 0.062, ± 0.0015 μmol min? 1 mg? 1 and 0.0019, ± 0.00064 μmol min? 1 mg? 1 for EA and BSP, respectively. Finally, the activity of the E-GST with the EA and BSP substrates, was measured by HPLC-PDA, and was found to be 0.027, ± 0.003 μmol min? 1 mg? 1 and 0.002, ± 0.0005 μmol min? 1 mg? 1, respectively. While the HPLC-PDA data do not mirror the spectrophotometric results quantitatively the overall response by the E-GST was the same. In general, the E-GSTs were shown to belong to the π-class when characterized by HPLC-PDA due to an EA specific activity greater than 0.01 μmol min? 1 mg? 1 and a negligible BSP activity (≤ 0.002 μmol min? 1 mg? 1). |
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