Dhica

Acetonitrile, creatinine, VMA, HVA, Trp, 5-HIAA and indoxyl sulphate (IS) were purchased from Sigma–Aldrich (St. Louis, MO, USA), DHICA from Toronto Research Chemicals Inc. (Toronto, ON, Canada), formic acid from Riedel-de Haёn (Seelze, Germany). Ultrapure water was prepared by ultrafiltration of distilled water using the Simplicity system (Millipore, Molsheim, France). Mobile phase of 15% Acetonitrile (ACN:H₂O; 15:85) was prepared in deionised water with an addition of 0.05% formic acid. Stock solutions of creatinine, DHICA, VMA, HVA, Trp, 5-HIAA and IS were prepared by diluting given compounds to a concentration of 1 mg/ml in mobile phase. All reagents were of analytical grade.

Acetonitrile (ACN), creatinine, IS, KYNA, Kyn, Trp, and 5-HIAA were purchased from Sigma–Aldrich (St. Louis, MO, USA), DHICA from Toronto Research Chemicals Inc. (Toronto, ON, Canada), formic acid from Riedel-de Haën (Seelze, Germany). Ultrapure water was prepared by ultrafiltration of distilled water using the Simplicity system (Millipore, Molsheim, France). Mobile phase of 15% Acetonitrile (ACN:H₂O; 15:85) was prepared in deionized water with an addition of 0.05% formic acid. Stock solutions of creatinine, DHICA, IS, KYNA, Kyn, Trp, and 5-HIAA were prepared by diluting given compounds to a concentration of 1 mg/mL in mobile phase. All reagents were of analytical grade.

TYRP1 (1.0 mg/mL) was reacted with 1.5 mM DHICA (Toronto Research Chemicals, Toronto, ON, Canada) that was diluted in buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl) at pH 5.5, 7.2, and 7.4. The different conditions of buffer for the experiments included the addition of 25 $\mathsf{\mu }$ M TCEP and/or 1.5 mM MBTH at each pH. This reaction was measured every hour for 4 h at 37 °C, shaking at 270 RPM. Additionally, TYRP1 (1.0 mg/mL) was only reacted with 1.5 mM DHICA that was diluted in buffer at pH 7.4 for 24 h at 37 °C shaking at 270 RPM. The spectrum from 200–900 nm was checked after the incubation on the NanoPhotometer N60-Touch, and the reaction was completed in triplicates, and measurements were averaged. The product of the reaction involving MBTH was measured at the peak of absorbance at 505 nm. The product of the ‘native’ reaction involving only TYRP1 and DHICA, IQCA, was measured at the absorbance peak at 560 nm.