Highly Amplified Spectrophotometry of Polyphenols Based on a Cyclic Reaction Between Polyphenols ando-Quinone Compounds Using Tyrosinase and L-Ascorbic Acid

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Abstract
Highly sensitive spectrophotometry for polyphenols has been proposed based on amplified pH changes during the substrate recycling of polyphenols driven by tyrosinase and L-ascorbic acid, which can be monitored as a spectral change in BTB (bromothymol blue). Polyphenolic compounds are enzymatically oxidized to o-quinone compounds catalyzed by tyrosinase, and are chemically regenerated by L-ascorbic acid, followed by substrate recycling. During this cyclic redox reaction, the pH continuously increases because a proton in the solution is consumed when o-quinones react with L-ascorbic acid. The magnitude of this pH change is significantly dependent on the concentration of the polyphenols, so a highly sensitive determination was possible by monitoring the absorbance of BTB in the enzyme solution with a reaction time of 5-10 min. The detection limits of catechol and catecholamines (L-dopa, dopamine, noradrenaline and adrenaline) were found to be on the order of 1x10−7 M-1x10−9 M using tyrosinase (catechol oxidase activity; 350 U ml−1), 1x10−2 M L-ascorbic acid and 0.005 % BTB.