Catechol O-methyltransferase plays an important role in the catabolism of catecholamine neurotransmitters such as dopamine, norepinephrine, and epinephrine, and inactivation of catechol estrogens and catechol xenobiotics. Several different methods have been developed.
In one, S-adenosyl-l-methionine was the methyl donor, norepinephrine was the substrate, while products of the reaction, normefanephrine and nor/wanephrine, were converted to the more stable and more easily obtained compounds vanillin and isovanillin, respectively, by periodate oxidation. The oxidation also allowed for the extraction of the incubation mixture with organic solvents such as ethyl acetate, affording a more complete deproteinization.
In this study (not shown), the compounds vanillin and isovanillin together with p-hydroxyacetanilide, added as an internal standard, were separated by reversed-phase HPLC (LiChrosorb) with a methanol-50 mM phosphate buffer (pH 7.2) (3:7, v/v) as the mobile phase. The compounds were eluted isocratically and the eluent monitored by an electrochemical detector.
In another study, the substrate was 3,4-dihydroxybenzoic acid, and the reaction products were 3-methoxy-4-hydroxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid. The substrate and the two products were separated by HPLC on a reversed-phase column (LiChrosorb) with a mobile phase of 0.05 M acetic acid in methanol-water (1:4, v/v), pH 3.2 Figure 9.12 shows the separation obtained under these conditions.
The reaction mixture was contained in a volume of 1 mL Tris-HCl buffer (pH 7.9), 5-adenosylmethionine, MgCl2,3,4-dihydroxybenzoic acid, and dithi-othreitol. Reactions were started by the addition of the activity and terminated
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