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ID 117346
Author
Mine, Masanori Tokushima University
Keywords
Capillary electrophoresis
Dynamic frontal analysis
Tyrosinase
Two-steps oxidation
Kinetic analysis
Content Type
Journal Article
Description
Tyrosinase catalyzes the oxidation of L-tyrosine in two stages to produce L-dopa and L-dopaquinone stepwise, and L-dopaquinone is subsequently converted to dopachrome. Most of the conventional analyses subjected only one-step reaction from L-tyrosine to L-dopa or from L-dopa to L-dopaquinone. In this study, kinetic analyses of two-steps oxidation of L-tyrosine with tyrosinase were made by capillary electrophoresis/dynamic frontal analysis (CE/DFA). When L-dopa was introduced into a capillary as a sample plug in a CE/DFA format, the enzymatic oxidation continuously occurred during the electrophoresis, and the product L-dopaquinone was subsequently converted to dopachrome which was detected as a plateau signal. A Michaelis-Menten constant of the second-step kinetic reaction, Km,Do, was determined as 0.45±0.03 mmol L−1. In the analysis of the first-step kinetic reaction from L-tyrosine to L-dopa, L-dopa was not resolved by CE/DFA because both L-tyrosine and L-dopa are electrically neutral. The L-dopa formed and co-migrated at the L-tyrosine zone was calibrated beforehand with the final product of dopachrome detected as a plateau signal. Constantly formed L-dopa was successfully detected as a plateau signal of dopachrome, and a Michaelis-Menten constant of Km,Ty was also determined as 0.061±0.009 mmol L−1 by the CE/DFA. CE/DFA is applicable to two-steps enzymatic reactions.
Journal Title
Analytical Biochemistry
ISSN
00032697
NCID
AA00524867
AA11537838
Publisher
Elsevier
Volume
655
Start Page
114856
Published Date
2022-08-11
Rights
© 2022. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/
EDB ID
DOI (Published Version)
URL ( Publisher's Version )
FullText File
language
eng
TextVersion
Author
departments
Science and Technology