Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid
Wimalasena, Kandatege Wimalasena, D. Shyamali
Wimalasena, Kandatege
Wimalasena, D. Shyamali
Location
Time Period
Advisors
Original Date
Digitization Date
Issue Date
1991-09-02
Type
Article
Genre
Keywords
Research Support, Non-U.S. Gov't
Subjects (LCSH)
Ascorbic Acid/chemistry
Citation
Analytical biochemistry. 1991 Sep 2; 197(2): 353-61.
Abstract
Based on the novel chromophoric electron donors, N,N-dimethyl-1,4-phenylenediamine (DMPD) and 2-amino-2-deoxy-L-ascorbic acid (2-aminoascorbic acid), two sensitive, convenient, and continuous spectrophotometric assays for dopamine beta-monooxygenase (EC 1.14.17.1) are described. Both, DMPD and 2-aminoascorbic acid are kinetically and stoichiometrically well-behaved electron donors for dopamine beta-monooxygenase with kinetic parameters comparable to the most efficient physiological electron donor, ascorbic acid. During dopamine beta-monooxygenase turnover, DMPD is converted to its chromophoric cation radical which is stable under the standard assay conditions. The rate of the enzyme-dependent formation of DMPD cation radical under standard assay conditions could easily be followed at 515 nm with high accuracy and reproducibility. Similarly, dopamine beta-monooxygenase-mediated oxidation of 2-aminoascorbic acid results in the formation of the known, stable chromophoric product, 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment), which has a very strong absorption maximum at 385 nm. Both the above assays are superior to the existing assays in their convenience, reproducibility, and sensitivity for routine kinetic analysis of dopamine beta-monooxygenase and may be adopted as a simple color test for the enzyme. We propose that the above assays could also be adopted to design continuous and sensitive spectrophotometric assays for ascorbate oxidase, peptidyl alpha-amidating monooxygenase, and the chromaffin granule electron transport protein, cytochrome b561, due to their remarkable similarity to dopamine beta-monooxygenase in the chemistry of catalysis with regard to the electron donor.
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Full text of this article is not available in SOAR.
Publisher
Elsevier
Journal
Book Title
Series
Analytical biochemistry
Anal. Biochem.
Anal. Biochem.
Digital Collection
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Archival Collection
NLM
PubMed ID
DOI
ISSN
0003-2697