A new continuous spectrophotometric assay method for DOPA oxidase activity of tyrosinase.
Sensitive assay methods for tyrosinase are essential not only for the understanding the process of pigment production but also for the development of effective inhibitors of tyrosinase. To develop an efficient assay method, we applied thymol blue to reaction mixtures. The enzyme kinetic study revealed that DOPA oxidase activity of tyrosinase in thymol blue-applied reaction system was more sensitively measured, even under lower enzyme units compared with the previous report with significant enhancement of Vmax while affinity change on substrate was not observed. To test whether this method could be applicable to the inhibition and the inactivation kinetic study of tyrosinase, the effect of kojic acid, a well-known tyrosinase inhibitor, and sodium chloride respectively, have been studied. Conclusively, thymol blue method can assay tyrosinase activity with sensitivity and is applicable to the inhibition and the inactivation study of tyrosinase.
Park
Yong-Doo
YD
Clinical Research Center, Samsung Biomedical Research Institute, Seoul 135-710, Korea.
Lee
Jae-Rin
JR
Park
Kyung-Hee
KH
Hahn
Hwa-Sun
HS
Hahn
Myong-Joon
MJ
Yang
Jun-Mo
JM
D000363:Agaricales / Q000201:enzymology; D002021:Buffers; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology; D011753:Pyrones / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013053:Spectrophotometry / Q000379:methods*; D013944:Thymolphthalein / Q000031:analogs & derivatives* / Q000494:pharmacology
D002021:Buffers; D010710:Phosphates; D011753:Pyrones; D012965:Sodium Chloride; C011890:kojic acid; C013713:thymol blue; D014442:Monophenol Monooxygenase; C018279:sodium phosphate; D013944:Thymolphthalein