In the literature, the polyphenol oxidase (PPO) enzyme has been purified a many times via Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. In order to study PPO purification efficiency, 2-aminophenol and 4-aminophenol were applied as a spacer arm to CNBr-activated Sepharose 4B. The effects of the spacer arm on specific activity, purification fold, and electrophoretic properties were investigated. The best performance with 11.7-fold purification and 23951 U/mg protein specific activity was achieved with the 4-aminophenol extension arm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with was done to check the purity of the potato PPO enzyme obtained from affinity columns. According to the results of SDS-PAGE and native PAGE, the molecular weight of the enzyme is 50 kDa. Furthermore, the inhibition effects of curcumin and quercetin on the enzyme activity were examined, and the IC50 and Ki values were computed for the mentioned substances. IC50 values were determined to be 0.018 and 0.029 mM for potato PPO with curcumin and quercetin inhibitors with catechol as a substrate, respectively. IC50 value was also determined to be 0.0086 mM for quercetin inhibitor with 4-methylcatechol as a substrate. Ki constant was 0.0753 ± 0.0085 mM for curcumin using catechol as a substrate. No inhibition effect was observed for curcumin with the 4-methylcatechol substrate. The Ki constant for quercetin was 0.0398 ± 0.00743 mM with the 4-methylcatechol substrate and 0.0109 ± 0.0021 mM with the catechol substrate.

中文翻译：

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对多酚氧化酶的纯化以及姜黄素和槲皮素对马铃薯多酚氧化酶的抑制作用的新见解。

在文献中，多酚氧化酶（PPO）酶已通过Sepharose 4B-1-酪氨酸-对氨基苯甲酸亲和柱纯化了许多次。为了研究PPO的纯化效率，将2-氨基苯酚和4-氨基苯酚作为间隔臂应用于CNBr活化的Sepharose 4B。研究了间隔臂对比活性，纯化倍数和电泳性能的影响。用4-氨基苯酚延伸臂可获得11.7倍纯化和23951 U / mg蛋白比活性的最佳性能。用十二烷基硫酸钠聚丙烯酰胺钠凝胶电泳（SDS-PAGE）检查从亲和柱获得的马铃薯PPO酶的纯度。根据SDS-PAGE和天然PAGE的结果，该酶的分子量为50kDa。此外，研究了姜黄素和槲皮素对酶活性的抑制作用，并计算了上述物质的IC50和Ki值。以姜黄素和槲皮素抑制剂（以邻苯二酚为底物）的马铃薯PPO的IC50值分别确定为0.018和0.029 mM。对于以4-甲基邻苯二酚为底物的槲皮素抑制剂，IC50值也确定为0.0086 mM。以儿茶酚为底物的姜黄素的Ki常数为0.0753±0.0085 mM。姜黄素与4-甲基邻苯二酚底物没有观察到抑制作用。槲皮素的Ki常数在4-甲基邻苯二酚底物下为0.0398±0.00743 mM，在邻苯二酚底物下为0.0109±0.0021 mM。以姜黄素和槲皮素抑制剂（以邻苯二酚为底物）的马铃薯PPO的IC50值分别确定为0.018和0.029 mM。对于以4-甲基邻苯二酚为底物的槲皮素抑制剂，IC50值也确定为0.0086 mM。以儿茶酚为底物的姜黄素的Ki常数为0.0753±0.0085 mM。姜黄素与4-甲基邻苯二酚底物没有观察到抑制作用。槲皮素的Ki常数在4-甲基邻苯二酚底物下为0.0398±0.00743 mM，在邻苯二酚底物下为0.0109±0.0021 mM。以姜黄素和槲皮素抑制剂（以邻苯二酚为底物）的马铃薯PPO的IC50值分别确定为0.018和0.029 mM。对于以4-甲基邻苯二酚为底物的槲皮素抑制剂，IC50值也确定为0.0086 mM。以儿茶酚为底物的姜黄素的Ki常数为0.0753±0.0085 mM。姜黄素与4-甲基邻苯二酚底物没有观察到抑制作用。槲皮素的Ki常数在4-甲基邻苯二酚底物下为0.0398±0.00743 mM，在邻苯二酚底物下为0.0109±0.0021 mM。使用儿茶酚作为底物的姜黄素为0085 mM。姜黄素与4-甲基邻苯二酚底物没有观察到抑制作用。槲皮素的Ki常数在4-甲基邻苯二酚底物下为0.0398±0.00743 mM，在邻苯二酚底物下为0.0109±0.0021 mM。使用儿茶酚作为底物的姜黄素为0085 mM。姜黄素与4-甲基邻苯二酚底物没有观察到抑制作用。槲皮素的Ki常数在4-甲基邻苯二酚底物下为0.0398±0.00743 mM，在邻苯二酚底物下为0.0109±0.0021 mM。