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Synchronous fluorometric method for continuous assay of monophenolase activity.

Ling Zhang1, Qi Shang1, Chan Chen1

  • 1Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, China.

Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
|January 31, 2021
PubMed
Summary

A new continuous assay method accurately measures tyrosinase monophenolase activity using synchronous fluorescence. This method enables efficient kinetic studies and inhibitor screening for tyrosinase, a key enzyme in melanogenesis.

Keywords:
BorateInhibitorMonophenolaseSynchronous fluorescenceTyrosine

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Area of Science:

  • Biochemistry
  • Enzymology
  • Analytical Chemistry

Background:

  • Tyrosinase is the crucial enzyme in melanogenesis, possessing both monophenolase and diphenolase activities.
  • Accurate measurement of tyrosinase's monophenolase activity is essential for understanding melanin production and for drug discovery.
  • Existing methods may not efficiently distinguish or quantify monophenolase activity in the presence of diphenolase activity.

Purpose of the Study:

  • To develop a continuous, selective assay for monitoring tyrosinase monophenolase activity.
  • To utilize synchronous fluorescence spectroscopy for sensitive detection of tyrosine consumption.
  • To apply the assay for kinetic analysis and inhibitor screening of tyrosinase.

Main Methods:

  • Developed a continuous assay employing synchronous fluorescence to monitor tyrosine hydroxylation.
  • Utilized borate complexation to quench DOPA fluorescence and selectively quantify tyrosine.
  • Employed hydroxylamine to maintain the enzyme's catalytic cycle and enable continuous monitoring.

Main Results:

  • Achieved a limit of detection (LOD) of 0.49 μM for tyrosine.
  • Established a calibration curve for tyrosinase concentration with an LOD of 0.0721 U·mL⁻¹.
  • Determined kinetic parameters (Km = 20.73 μM, Vmax = 1.10 μM·min⁻¹) and identified zinc ion as a competitive inhibitor (IC50 = 14.36 μM).

Conclusions:

  • The developed synchronous fluorescence assay provides a robust method for direct measurement of tyrosinase monophenolase activity.
  • This assay is effective for enzyme kinetics studies and high-throughput screening of tyrosinase inhibitors.
  • The method offers a valuable tool for research in melanogenesis and related dermatological conditions.