ZÍTKA Ondřej1, KRYŠTOFOVÁ Olga1, CERNEI Natalia1, ADAM Vojtěch1,
HUBÁLEK Jaromír1, TRNKOVÁ Libuše2, BEKLOVÁ Miroslava3, KIZEK René Kizek1
(1Mendelova univerzita v Brně, 2Masarykova univerzita, 3Veterinární a farmaceutická univerzita v Brně)
Vysoce účinná kapalinová chromatografie jako nástroj pro detekci fytochelatin syntázy High performance liquid chromatographic assay for detection of phytochelatinsynthase
Phytochelatins are sulphur-rich peptides able to detoxify heavy metals. Their synthesis is catalyzed by the enzyme called
phytochelatin synthase. In this study, we used high performance liquid chromatography with electrochemical detection (HPLC-ED)
for sensitive determination of phytochelatins and phytochelatin synthase activity. Chromatographic conditions were optimized.
Phytochelatin synthase activity was determined in homogenate prepared from cell suspension cultures of tobacco
(Nicotiana tabacum cv. BY-2). The enzyme was induced by 50 µM Cd(NO3)2. As a substrate, we used
100 mM reduced glutathione (GSH).
Enzyme activity was determined as the equivalent of synthesized phytochelatins after an incubation lasting 15 minutes.
Phytochaltins were detected in the reaction mixture by HPLC-ED. The results obtained show that the optimized HPLC-ED method is
suitable not only for detection of low concentrations of phytochelatins, but also for determination the activity
of phytochelatin synthase.
Key words: glutathione, phytochelatins, phytochelatin synthase, high performance liquid chromatography with electrochemical detection (HPLC-ED).