| Tyrosinase(TYR)(monophenol or diphenol,oxidase,EC 1.14.18.1)is a multifunctional,glycosylated,copper-containing oxidase of melanocyte that is found primarily in specialized organelles called melanosomes.TYR is widely distributed in bacteria,plants and mammals.So far,there has been a large amount of evidence that TYR plays a key role in the physiological processes related to tyrosine,such as plants browning reaction,insect cuticle sclerotization,oxidative stress and the generation of melanin in mammals.For example,in the presence of molecular oxygen,TYR catalyzed o-hydroxylation of monophenol to form o-diphenol,and further oxidation of o-diphenol forms o-quinone,promoting the synthesis of melanin in melanocytes.It has been reported that the activity or level of TYR directly affects the generation of melanin,which is closely related to vitiligo,melanoma and other serious skin diseases.Tyrosinase is a key enzyme for the biosynthesis of melanin,which is overexpressed in melanoma cells.Therefore,tyrosinase is also considered as a biomarker and therapeutic target for vitiligo,melanoma and other severe skin diseases.The traditional methods for detecting TYR mainly include colorimetry,high performance liquid chromatography(HPLC),immunoassays,spectrophotometry,and electrochemical detection,etc.These methods have complicated sample preparation,poor biocompatibility,and are not suitable for real-time detection.What’s more,the results detected by these methods are significant different.These methods are not suitable for accurate detection.Compared with the traditional detection methods,fluorescence detection method has the advantages of low detection cost,simple operation,good selectivity,high sensitivity,and good biocompatibility.It can be used for real-time detection of many biological reactive species in cells and living organisms.In this work,a turn-on fluorescent probe W-NBD capable of rapidly detecting TYR activity was designed and synthesized by using 3-hydroxybenzylamine as the recognition group of TYR.It is based on native chemical ligation(intramolecular S→N conversion).The experimental results show that the probe can selectively detect TYR in PBS buffer solution.The selective detection of TYR solves the problem that the reported probes have been interfered with reactive oxygen species,and the probe has also been used to detect the activity of TYR in the whole protein extract of cells,showing a good linear relationship.Finally,based on the probe W-NBD,we designed and synthesized a ratiometric fluorescent probe B-BODIPY,which can rapidly and selectively detect TYR activity in buffer solution. |
