Využití diferenční skenovací fluorimetrie v charakterizaci vybrané karbonylreduktasy

• Main Author: Tomanová, Radana
• Other Authors: Zemanová, Lucie
• Format: Dissertation
• Language: Czech
• Published: 2012
• Online Access: http://www.nusl.cz/ntk/nusl-307788

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Radana Tomanová Supervisor: RNDr. Lucie Škarydová, Ph.D. Title of diploma thesis: The use of differential scanning fluorimetry in characterization of selected carbonyl reductase Differential scanning fluorimetry (DSF) is simple, rapid method that enables to determine optimal conditions for stabilization of proteins and discover their ligands. DSF monitors thermal unfolding of a protein in the presence of fluorescent dye (e.g. SYPRO Orange, 2,6-ANS). The dye is highly fluorescent in non-polar environment such as hydrophobic sites of unfolded protein that appear on the surface in gradual increase of temperature. Melting temperature (Tm) of a protein expresses its stability. Ligand screening relies upon the fact that protein stability is enhanced upon ligand binding (ΔTm  0). The aim of this study was to introduce the DSF method on our department, use it for characterization of carbonyl reductase 1 (CBR1) and evaluate the obtained results with an independent methods and literature. First, the functionality of method was verified with citrate synthase and the conditions were optimized for CBR1. The group of several buffers pH 3-10 without additives or with NaCl or glycerol were tested...