Institute of Chemistry and Biochemistry, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic.
J Plant Physiol. 2012 Sep 1;169(13):1317-20. doi: 10.1016/j.jplph.2012.04.008. Epub 2012 Jun 6.
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.
大量研究已经估计了苯丙氨酸解氨酶（PAL）的活性，因为它对各种刺激强烈反应。这种酶的活性主要通过分光光度法来测定，但这种方法的精度知之甚少。我们在两种植物（母菊和拟南芥）中比较了使用分光光度法和高效液相色谱法（HPLC）测定 PAL 活性。此外，还测试了暴露在铜中的母菊植物和含有添加剂的缓冲液。我们的数据表明，与 HPLC 相比，分光光度法高估了 PAL 活性（母菊的叶片）和低估了 PAL 活性（拟南芥的叶片和根），这表明 UV 吸收代谢物的干扰。HPLC 还显示了在铜暴露的母菊根中更准确地检测肉桂酸。向提取缓冲液中添加二硫苏糖醇会增强 PAL 活性但降低蛋白质，表明存在人为的负面影响。还提供了所选物种中 PAL 活性的比较。
