Cu²⁺ release and inactivation of tyrosinase are mediated by 1MHz ultrasound

Abstract

[Purpose] In recent works, sonotherapy have been expected to apply extensively to dermatological disorder and skin homeostasis. Tyrosinase is one of target enzymes of sonotherapy for melanin deposition. In this report, we tried to inactivate tyrosinase in vitro by 1 MHZ ultrasound and to elucidate the basic mechanism of sonotherapy of skin. [Method] As an enzymatic assay of tyrosinase, continuous spectrophotometric rate determination was employed. Reaction mixture (50 nM L-dopa, 2 % DMF, 6 nM MBTH in 1 mM phosphate buffer pH 7.1) containing Mushroom tyrosinase (1 kunits/mg) was prepared and used for the assay. Reactive oxygen species (ROS) were detected by the method combining ESR and spin-trapping. Released Cu²⁺ was quantified with kinetic-catalytic spectrophotometric method. Sonication was performed with 1 MHz Ultax-UX601 (-1.4 W/cm²). [Results] Tyrosinase activity was reduced as increasing power of 1 MHz ultrasound. When the reaction mixture containing spin trap was irradiated, OH-adduct was detected by ESR. However, there was no effect of mannitol (OH scavenger) on the reduction of tyrosinase activity after irradiation. When excess Cu²⁺ was added to the solution, tyrosinase activity was not reduced after irradiation. The analysis of free Cu²⁺ showed that ultrasound mediated Cu²⁺ release from tyrosinase. These results indicate that ultrasound-induced inactivation of tyrosinase is caused by not ROS generated from the cavitaion but the Cu²⁺ release from activation center of the enzyme.