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Development of universal affinity matrix for isolation of carbonyl reducing enzymes from natural samples
Authors: Andrýs Rudolf | Škarydová Lucie | Holubová Lucie | Bílková Zuzana | Wsól Vladimír
Year: 2012
Type of publication: ostatní - přednáška nebo poster
Page from-to: nestránkováno
Titles:
Language Name Abstract Keywords
eng Development of universal affinity matrix for isolation of carbonyl reducing enzymes from natural samples Carbonyl reducing enzymes play an important role in metabolic pathways of various eobiotics and xenobiotics e.g. steroids, prostaglandins, doxorubicin, daunorubicin etc. Moreover, they contribute to development of some diseases like hormone-dependent cancers, metabolic syndrome or Cushing’s syndrome. In spite of this fact there is still little known about them. Some of these enzymes have been only poorly characterized or totally uncharacterized yet. Due to their importance, it is necessary to make further studies on their activity, properties and distribution. Unfortunately, carbonyl reducing enzymes are in tissues usually in quite low concentrations so their isolation with classical techniques is complicated. It is difficult to obtain fraction with sufficient purity and amount of desired enzyme. The aim of the study is the preparation of an affinity matrix for selective isolation of carbonyl reducing enzymes from natural samples. Oracin is a potential anticancer drug that has been shown as a substrate for many well known carbonyl reducing enzymes so it seems to be universal ligand for development of the affinity matrix. Four types of magnetic microparticles, SiMAG-NH2, SiMAG-COOH, SiMAG-PGL and magnetic macroporous bead cellulose have been used as carriers. The molecule of oracin have been modified and bound in different ways to carriers. Optimal binding method and carrier was chosen for further studies. The ability of prepared affinity matrix to capture carbonyl reducing enzymes was tested on model enzymes AKR1C3 and CBR1. Enzymes were selectively bound on affinity matrix and then eluted. It is clear that the new affinity matrix is well working with reductases AKR1C3 and CBR1 and it is ready for use in isolation process of carbonyl reducing enzymes from natural samples. This project was supported by the Grant Agency of Charles University (Grant no. 71710/C/2010) and also by the Charles University in Prague (the SVV 265 004) and the project UNCE No. (17/2012). affinity chromatography, carbonyl-reducing enzymes, oracin, magnetic particles

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