Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.)
Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.)
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DOI：
10.1021/jf049053g
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发表时间：
2005-02-09
影响因子：
6.1
通讯作者：
Arslan, O
中科院分区：
文献类型：
--
作者：
Dogan, S;Turan, Y;Arslan, O
在本研究中，首先通过 (NH4)(2)SO4 沉淀、透析和 Sepharose 4B-L-酪氨酸-对氨基苯甲酸亲和柱的组合纯化朝鲜蓟 (Cynara scolymus L.) 的多酚氧化酶 (PPO)。纯化结束时，实现了43倍纯化。纯化的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上迁移为单条带。聚丙烯酰胺凝胶电泳表明PPO的分子量为57 kDa。其次，测定了朝鲜蓟头提取物中总酚和蛋白质的含量。根据Folin-Ciocalteu程序，用分光光度法测定朝鲜蓟头的总酚含量，发现以鲜重计为425mg·100g(-1)。蛋白质含量按照Bradford法测定。第三，研究了底物特异性、pH、温度和热灭活对从朝鲜蓟中纯化的 PPO 活性的影响。该酶对 4-甲基儿茶酚、连苯三酚、儿茶酚和 L-多巴具有活性。未检测到 L-酪氨酸、间苯二酚和对甲酚的活性。根据V-max/K-m值，4-甲基儿茶酚(1393 EU min(-1) mM(-1))是最好的底物，其次是邻苯三酚(1220 EU min(-1) mM(-1))、儿茶酚(697 EU min(-1) mM(-1))和L-多巴(102 EU min(-1) mM(-1))。以 4-甲基儿茶酚、邻苯三酚和邻苯二酚为底物时，PPO 的最适 pH 值分别为 5.0、8.0 和 7.0。结果发现，最佳温度取决于所研究的基材。随着温度的升高和 4-甲基儿茶酚和连苯三酚底物失活时间的增加，酶的热变性导致酶活性降低。然而，所有儿茶酚的灭活实验都表明，朝鲜蓟PPO的活性随着温和加热而增加，达到最大值，然后随着时间的推移而降低。最后，以 4-甲基儿茶酚、连苯三酚和儿茶酚为底物，研究了 L-半胱氨酸、EDTA、抗坏血酸、没食子酸、D,L-二硫苏糖醇、托酚酮、谷胱甘肽、叠氮化钠、苯甲酸、水杨酸和 4-氨基苯甲酸等抑制剂对朝鲜蓟 PPO 的抑制作用。 EDTA、4-氨基苯甲酸、水杨酸、没食子酸和苯甲酸的存在不会引起朝鲜蓟PPO的抑制。以4-甲基儿茶酚为底物，使用叠氮化钠、L-半胱氨酸和D,L-二硫苏糖醇抑制剂获得竞争型抑制；与 L-半胱氨酸、托酚酮、D,L-二硫苏糖醇、抗坏血酸和叠氮化钠抑制剂一起使用连苯三酚作为底物；以及使用儿茶酚作为底物的 L-半胱氨酸、托酚酮、D,L-二硫苏糖醇和抗坏血酸抑制剂。使用 4-甲基儿茶酚作为底物，用谷胱甘肽抑制剂获得了混合型抑制。以4-甲基儿茶酚为底物的托酚酮和抗坏血酸抑制剂、以连苯三酚为底物的谷胱甘肽抑制剂以及以儿茶酚为底物的谷胱甘肽和叠氮化钠抑制剂均获得非竞争性抑制。从这些结果可以说，朝鲜蓟PPO最有效的抑制剂是托酚酮。此外，还发现抑制的类型取决于所研究的 PPO 的来源以及所使用的底物。
In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH4)(2)SO4 precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V-max/K-m values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic acid, gallic acid, D,L-dithiothreitol, tropolone, glutathione, sodium azide, benzoic acid, salicylic acid, and 4-aminobenzoic acid using 4-methylcatechol, pyrogallol, and catechol as substrate. The presence of EDTA, 4-aminobenzoic acid, salicylic acid, gallic acid, and benzoic acid did not cause the inhibition of artichoke PPO. A competitive-type inhibition was obtained with sodium azide, L-cysteine, and D,L-dithiothreitol inhibitors using 4-methylcatechol as substrate; with L-cysteine, tropolone, D,L-dithiothreitol, ascorbic acid, and sodium azide inhibitors using pyrogallol as substrate; and with L-cysteine, tropolone, D,L-dithiotreitol, and ascorbic acid inhibitors using catechol as a substrate. A mixed-type inhibition was obtained with glutathione inhibitor using 4-methylcatechol as a substrate. A noncompetitive inhibition was obtained with tropolone and ascorbic acid inhibitors using 4-methylcatechol as substrate, with glutathione inhibitor using pyrogallol as substrate, and with glutathione and sodium azide inhibitors using catechol as substrate. From these results, it can be said that the most effective inhibitor for artichoke PPO is tropolone. Furthermore, it was found that the type of inhibition depended on the origin of the PPO studied and also on the substrate used.