Tissue and method specificities of phenylalanine ammonia-lyase assay.
A large number of studies have estimated phenylalanine ammonia-lyase (PAL) activity because it strongly reacts to various stimuli. Activity of this enzyme has been assayed mainly by means of spectrophotometry, but the precision of this method is poorly known. We compared assays of PAL activity using spectrophotometry and high performance liquid chromatography (HPLC) in two species (Matricaria chamomilla and Arabidopsis thaliana). Additionally, copper-exposed M. chamomilla plants and buffer with additive were also tested. Our data indicate that spectrophotometry both overestimates (leaves of M. chamomilla) and underestimates (leaves and roots of A. thaliana) PAL activity in comparison with HPLC, suggesting interference of UV-absorbing metabolites. HPLC also showed more accurate detection of cinnamic acid in Cu-exposed chamomile roots. Addition of dithiothreitol to the extraction buffer enhanced PAL activity but reduced proteins, indicating an artificial negative effect. A comparison of PAL activity in selected species is also provided.
Institute of Chemistry and Biochemistry, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic. jozkovacik@yahoo.com
Jozef
J
Kováčik
Bořivoj
B
Klejdus
D017360:Arabidopsis / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000378:metabolism; D031168:Matricaria / Q000201:enzymology*; D010636:Phenols / Q000378:metabolism*; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D018517:Plant Roots / Q000201:enzymology; D013053:Spectrophotometry; D013379:Substrate Specificity
D010636:Phenols; D003300:Copper; D010650:Phenylalanine Ammonia-Lyase