Tyrosine hydroxylase is a monooxygenase that catalyzes the conversion of l-tyrosine to Dopa. The activity is found in peripheral and cholinergic neurons and chromaffin cells of the adrenal medulla. HPLC methods have been developed for the assay of this activity.
In one method, the Dopa formed during the reaction was partially purified by ion-exchange and aluminum oxide chromatography and the amount present quantified by reversed-phase HPLC (ODS column). The mobile phase consisted of 0.1 M potassium phosphate buffer at pH 3.5. The column was eluted isocratically and the eluent monitored by means of an electrochemical detector.
The volume of the reaction mixture used with the HPLC assay was only about one-fifth the volume usually required with other assay methods, resulting in a considerable saving in reagents. The reaction was terminated with perchloric acid, the pH of the solution was returned to about 8 with potassium carbonate, and the sample was clarified by centrifugation. First the supernatant solution was purified using the double-column chromatographic procedure mentioned above, and then the samples were injected onto the HPLC column and analyzed for Dopa.
With the electrochemical detector, it is possible to quantitate the amount of Dopa present. The presence of pterins, cofactors required for activity, can also be detected.
Also, as noted, the HPLC method eliminates the need to know the concentration of tyrosine in the tissue. Such information would be required in radiochemical assays of activities, since any unlabeled substrate, in this case tyrosine, would reduce the specific activity of the labeled tyrosine.
In the second method, there was no prepurification: Dopa was measured directly. The separation of Dopa and tyrosine was carried out on a cationic ion-exchange HPLC (sulfonated fluorocarbon polymer) protected with a pre-column packed with silica pellets. The column was eluted with a mobile phase of 10 mM acetate buffer (pH 3.70) with 1% (v/v) propanol. The separation of tyrosine and Dopa is shown in Figure 9.1 A Because of its unique natural fluorescence, the Dopa can be monitored with a fluorescence detector without interference from endogenous substances. An excitation wavelength of 281 nm was used with the emission of 314 nm.
The reaction mixture contained the substrate L-tyrosine. Benzyloxyamine was added to inhibit any secondary reactions catalyzed by the enzyme aromatic L-amino acid decarboxylase, an activity often present in these samples. Chro-matograms of samples taken during an incubation are shown in Figure 9.IS, a zero-time control with a single peak of tyrosine, and Figure 9.1C, after 20 minutes of incubation showing the peak of Dopa formed as a result of enzymatic activity.
The assay described by Naoi et al. (1988) is sensitive enough to eliminate the need for the purification and concentration steps required in other procedures.
L-Dopa was separated on a Cosmosil 5 Qg column (4.6 mm x 250 mm). The mobile phase contained 90 mM sodium acetate, 35 mM citric acid, 130 ftM disodium EDTA, and 230 fiM sodium n-octanesulfonate in 10.5%
0123 0123 01234 Retention time (min)
Figure 9.1 (A) Chromatogram of 384 pmol of Dopa (1.03 min) and 1.92 nmol of l-tyrosine (1.55 min). (B) and (C) Chromatograms of an acidic ethanol extract of an incubation mixture in the assay of tyrosine 3-monoxygenase activity of bovine adrenal medulla microsomes. (B) Zero-time control with a single peak of l-tyrosine (1.55 min). (C) The formation of Dopa (1.03 min) following a reaction period of 20 minutes. Volumes of 20 ¡jlL of the diluted (twice) incubation mixture were injected into the liquid chromatograph. Detection wavelengths for excitation and emission were 281 and 314 nm, respectively. (From Haavik and Flatmark, 1980.)
methanol. The sample was eluted at a flow rate of 0.8 mL/min at room temperature. Detection and quantitation was by use of a Coulochem electrochemical detector. The voltage of a conditioning cell was set at +250 mV, and those of the first and second electrodes of an analytical cell were set at +50 and -300 mV, respectively. The output of the latter electrode was monitored. Quantitation of L-Dopa was carried out by comparison of the peak area with that of standards.
The reaction mixture contained in a total volume of 100 fiL: 200 fiM l-tyrosine, 100 mAf sodium acetate buffer (pH 6.0), 1 mM (6R)-L-erythro-5,6, 7,8-tetrahydrobiopterine or (67?5)-methyl-5,6,7,8-tetrahydropterin, 10 fig of catalase, and 1 mAf NSD-1055 (inhibitor of aromatic L-amino acid decarboxylase). The stock pterin solution were made up to be 10 mM in 1 M mercaptoeth-anol. Assays of brain homogenates also included 2 mM ferrous ammonium sulfate. The amount of the source of tyrosine hydroxylase was brain homoge-nate (500-700 /¿g protein) or PC12h cells (50 ¡ig protein). Reactions were started by adding tyrosine and the pterin cofactor. After incubation at 37°C for 10 minutes, the reaction was terminated by addition of 100 /¿L of 0.1 M perchloric acid, containing 0.4 mM sodium metabisulfite and 0.1 mM EDTA. The resulting mixture was allowed to stand on ice for 10 minutes before centrifugation and analysis of an aliquot of the supernate by HPLC. The reaction was linear from 5 to 200 fig of PC12h cell protein, and with time up to 10 minutes.
Brain samples were homogenized with 10 volumes of 10 mM phosphate buffer (pH 7.4). The homogenates were centrifuged through a Centricut centrifuge tube, which retained molecules of molecular mass less than 20,000. The retentate was assayed. A clonal rat pheochromocytoma cell line, PC12h, was suspended in 10 mM potassium phosphate buffer (pH 7.4) and homogenized by sonication.
In the assay described by Mandai et al. (1992), the product, l-Dopa, was separated on a TSK-ODS-120T column (4.6 mm X 250 mm). The mobile phase contained 10.5% methanol, 90 mM sodium acetate, 35 mM citric acid, 0.13 mM disodium EDTA, and 0.23 mM sodium n-octanesulfonate. The effluent was monitored by fluorescence using excitation and emission wavelengths of 281 and 314 nm, respectively.
The standard assay contained retinal homogenate (0.2-3 mg protein), 0.05 mM tyrosine, 1 mM (6/?,5)-5,6,7,8-tetrahydro-l-biopterin, 3.5 mM NADH, 0.02 unit of dihydroxypteridine reductase, 15 /ng catalase, 40 mM sodium acetate buffer (pH 6.0), and 0.1 mM o-benzylhydroxylamine (inhibitor of Dopa decarboxylase) in a final volume of 100 yiL. After incubation of the mixture at 37°C for 5 to 20 minutes, the reaction as stopped by adding 100 /xL of 0.5 M perchloric acid containing 0.4 mM sodium metabisulfite and 0.1 mM disodium EDTA. The supernate obtained after centrifugation was used for HPLC analysis.
The retina of bovine eye was homogenized with a Teflon homogenizer in one volume of Dulbecco's phosphate-buffered saline (PBS) containing protease inhibitors (4 /xg/mL of p-amidinophenylmethanesulfonylfluoride and 10 /xg/mL of leupeptin). The supernate obtained by centrifugation was passed through a Sephadex G-25 column before being used for assays.
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