HIGHLY AMPLIFIED SPECTROPHOTOMETRY OF POLYPHENOLS BASED ON A CYCLIC REACTION BETWEEN POLYPHENOLS AND O-QUINONE COMPOUNDS USING TYROSINASE AND L-ASCORBIC-ACID

Highly sensitive spectrophotometry for polyphenols has been proposed b ased on amplified pH changes during the substrate recycling of polyphe nols 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 tyrosi nase, and are chemically regenerated by L-ascorbic acid, followed by s ubstrate recycling. During this cyclic redox reaction, the pH continuo usly increases because a proton in the solution is consumed when o-qui nones react with L-ascorbic acid. The magnitude of this pH change is s ignificantly dependent on the concentration of the polyphenols, so a h ighly sensitive determination was possible by monitoring the absorbanc e of BTB in the enzyme solution with a reaction time of 5-10 min. The detection limits of catechol and catecholamines (L-dopa, dopamine, nor adrenaline and adrenaline) were found to be on the order of 1 x 10(-7) M-1 x 10(-9) M using tyrosinase (catechol oxidase activity; 350 U ml( -1)), 1 x 10(-2) M L-ascorbic acid and 0.005 % BTB.