Horseradish esterases: detection,purification and identification |
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Authors: | Ivana Le??i? A?ler Petra Peharec ?tefani? Biljana Balen Günter Allmaier Martina Marchetti-Deschmann Biserka Koji?-Prodi? |
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Institution: | 1.Department of Physical Chemistry, Laboratory for Chemical and Biological Crystallography,Ru?er Bo?kovi? Institute,Zagreb,Croatia;2.Division of Molecular Biology, Department of Biology, Faculty of Science,University of Zagreb,Zagreb,Croatia;3.Institute for Chemical Technologies and Analytics,Technische Universit?t Wien (Vienna University of Technology),Vienna,Austria |
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Abstract: | Our goal is to characterize esterases from horseradish tissues and assign their physiological roles. In the present study we focused on isolation, purification and identification of esterases from different horseradish tissues: plantlets and two tumor tissue lines. Horizontal IEF system enabled separation of six esterase isoforms with quite different pI values as well as with pronounced differences in expression levels among analyzed tissues. Esterases were extracted, fractionated by means of cation exchange chromatography, and analyzed by planar gel electrophoresis (SDS–PAGE) and isoelectrical focusing (IEF), UV/Vis spectroscopy, MALDI mass spectrometry (MS) and MALDI-MS/MS. Several chromatographic strategies were applied for esterase purification and characterization. Two subsequent cation exchange chromatographic steps based on SP-Sepharose FF material, followed by in-solution digestion combined with MALDI-MS and MS/MS proved to be the best strategy for identification of two esterase proteins, namely Pectinesterase/pectinesterase inhibitor 18 and GDSL esterase/lipase ESM1. |
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