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Real-time fluorometric monitoring of monophenolase activity using a matrix-matched calibration curve.
- Source :
-
Analytical and bioanalytical chemistry [Anal Bioanal Chem] 2021 Jan; Vol. 413 (2), pp. 635-647. Date of Electronic Publication: 2020 Nov 07. - Publication Year :
- 2021
-
Abstract
- Tyrosinase is the key enzyme for the metabolism of tyrosine and inherently comprises both monophenolase activity and diphenolase activity. A real-time fluorometric assay method was established to exclusively monitor the monophenolase activity by eliminating interference from diphenolase reactions through a combination of borate and hydroxylamine. Synthetic matrices comprised of tyrosine and DOPA (L-3,4-dihydroxyphenylalanine) preincubated with tyrosinase with the consistent sum concentration of 70 μM to mimic the monophenolase reaction mixture in borate buffer according to law of mass conservation. A matrix-matched calibration curve for determination of tyrosine was established using the synthetic matrices as standard sample to eliminate spectral interference from DOPA. The limit of detection (LOD) for tyrosine was 0.61 μM. The time course for consumption of tyrosine was established to measure the initial velocity through real-time reading out the tyrosine fluorescence intensity of the reaction mixture in a cuvette in situ. The assay worked in the monophenolase activity range from 0.2839 to 1.7308 U mL <superscript>-1</superscript> with LOD of 0.0851 U mL <superscript>-1</superscript> . The proposal sensing system successfully afforded a prospective potential for application in enzyme kinetics and screening of inhibitor. Graphical abstract.
- Subjects :
- Catalytic Domain
Chemistry Techniques, Analytical
Chromatography, High Pressure Liquid
Chromatography, Liquid
Enzyme Inhibitors chemistry
Kinetics
Limit of Detection
Monophenol Monooxygenase chemistry
Oxidation-Reduction
Reproducibility of Results
Time Factors
Calibration
Dihydroxyphenylalanine analysis
Fluorometry methods
Oxidoreductases analysis
Tyrosine analysis
Subjects
Details
- Language :
- English
- ISSN :
- 1618-2650
- Volume :
- 413
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Analytical and bioanalytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 33159571
- Full Text :
- https://doi.org/10.1007/s00216-020-03034-4