A Microcal VP-ITC system (Malvern Instruments) was used to conduct the ITC measurements as previously described.62 (link) The titration buffer consisted of 100 mM KPi with 50 mM NaCl at pH 8. L-tyrosine or analogs were prepared in titration buffer and injected to the cell containing 2 mL of 100–120 μM purified enzyme. Binding of L-tyrosine and its analogs was assessed in a total volume of 300 µL at the following concentrations: 1.5 mM 1 , 10 mM 2 and 20 mM 3 . After the temperature was equilibrated to 20 °C, a total of 50 injections were performed with a reference power of 15 μcal/s and a stirring speed of 350 rpm. The ITC data were processed and analyzed using non-linear least squares curve fitting of one-site binding models with Origin version 7.0 (OriginLab Corp.) software.
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Isothermal Titration Calorimetry of Tyrosine Binding
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Corresponding Organization : The University of Texas at San Antonio
Other organizations : Muhlenberg College
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Variable analysis
independent variables
- Ligand concentration: 1.5 mM for compound 1, 10 mM for compound 2, and 20 mM for compound 3
- Binding of L-tyrosine and its analogs
- Titration buffer: 100 mM KPi with 50 mM NaCl at pH 8
- Enzyme concentration: 100-120 μM purified enzyme
- Temperature: 20 °C
- Injection parameters: 50 injections, reference power of 15 μcal/s, stirring speed of 350 rpm
- No positive or negative controls were explicitly mentioned
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