Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid.
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.
Wimalasena
K
K
Department of Chemistry, Wichita State University, Kansas 67208.
Wimalasena
D S
DS
D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D002417:Cattle; D002624:Chemistry, Clinical; D002837:Chromaffin Granules / Q000201:enzymology; D004299:Dopamine beta-Hydroxylase / Q000032:analysis*; D007700:Kinetics; D010084:Oxidation-Reduction; D010655:Phenylenediamines / Q000737:chemistry; D013053:Spectrophotometry / Q000379:methods*
D010655:Phenylenediamines; C017945:scorbamic acid; C004781:dimethyl-4-phenylenediamine; D004299:Dopamine beta-Hydroxylase; D001205:Ascorbic Acid