A new insight into purification of polyphenol oxidase and inhibition effect of curcumin and quercetin on potato polyphenol oxidase.

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 K_i 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. K_i 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 K_i constant for quercetin was 0.0398 ± 0.00743 mM with the 4-methylcatechol substrate and 0.0109 ± 0.0021 mM with the catechol substrate.