PubMed · 5769774
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J Adams-Ray. 1969. Sunlight and catecholamines.. https://doi.org/10.1016/s0039-6109(16)38836-3
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The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% alpha-helix, 35% beta-extended structures (including beta-sheet), 14% beta-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in alpha-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.
An end-column electrical decoupler contructed with perfluorosulfonated ionomer (Nafion) is described. This decoupler was fabricated at the cathodic end of the separation capillary by casting liquid Nafion ion exchange powder over a copper-plated tungsten wire. The internal diameter of the flow channel was controlled by adjusting the thickness of the copper plating. This design overcomes problems of conventional end-column detection such as low sensitivity due to low collection efficiency of analytes at the detection electrode, difficulty in precise placement of the detection electrode, and the need to use small-bore capillaries (<-25 micron). The loss of cationic analytes observed with long-cast Nafion on-column decouplers was significantly reduced. The high current shunting capability of the long Nafion decoupler was maintained in this configuration. Elimination of the detection capillary required for on-column electrical decouplers provided higher separation efficiency by maintaining plug-type flow throughout the system. Four model catecholamines were well separated within 5 min with separation efficiency of up to 230 000 plates and migration time reproducibilities of <0.6% RSD. With the optimized experimental conditions, detection limits of 3 nM for the catecholamines were achieved in a Ringer's solution matrix (to model a microdialysis sample).
Using fused-silica optical fibres for fluorescence light collection and bandpass filters for selection of emission wavelengths, a capillary electrophoresis detection cell of a conventional, tunable UV-Vis absorbance detector was adapted for simultaneous fluorescence (at selected emission wavelength) and absorbance (at selected excitation wavelength) detection. Detector performance is demonstrated with the monitoring of underivatized fluorescent compounds in body fluids by micellar electrokinetic capillary chromatography with direct sample injection. Compared with UV absorption detection, fluorescence detection is shown to provide increased selectivity and for selected compounds also up to tenfold higher sensitivity. Examples studied include screening for urinary indole derivatives (tryptophan, 5-hydroxytryptophan, tyrosine, 3-indoxyl sulfate and 5-hydroxyindole-3-acetic acid) and catecholamine metabolites (homovanillic acid and vanillylmandelic acid) and the monitoring of naproxen in serum, quinidine in serum and urine and of salicylate and its metabolites in serum and urine.