Sodium ascorbate

For quantification of aromatic DHPAAS substrates and products, soluble DHPAAS (2–3 µg) in PBS was mixed with an aqueous solution of L-DOPA to a final volume of 40 µL. A final concentration of 1.875 mM L-DOPA was used together with 2.5 mM sodium ascorbate (Wako). Reactions were started at room temperature (23–24 °C) and transferred to 4 °C after 8 h. At various time points, 2 µL of each reaction was diluted into 98 uL methanol supplemented with ascorbate and camphor sulfonic acid standard. Dilutions were performed in triplicate. DHPAAS reaction dilutions were immediately stored at −30 °C until triplicate LC-MS analysis. In vitro data were analyzed with Prism 7.
H₂O₂ production was analyzed in 96 well plates using a fluorometric hydrogen peroxide assay kit (Sigma). 0.6–0.8 µg soluble DHPAAS in PBS (20 µL) was mixed with varying concentrations of L-DOPA (10 µL), followed by the addition of 30 µL peroxidase enzyme and fluorescent substrate mix (Sigma). Fluorescence was detected with a SpectraMax Paradigm microplate reader (Molecular Devices). Kinetic data included eight independent conditions for wild-type DHPAAS (one outlier), Asn192His variant (two outliers), and Phe79Tyr-Tyr80Phe variant, while 15 independent conditions were used for the Phe79Tyr-Tyr80Phe-Asn192His variant. Kinetic data was analyzed using the kcat function of Prism 7 with outlier elimination.

The following chemicals and materials were obtained from the indicated companies: Dulbecco’s modified Eagle’s medium (DMEM) from Gibco BRL, Grand Island, NY, USA; fetal bovine serum (FBS), benzaldehyde (MW = 106) (purity: >98%), sodium ascorbate (MW = 198), eugenol (MW = 164) (purity: >98%), NaF, D-mannitol, 20% glutaraldehyde solution, dimethylsulfoxide (DMSO) from Wako Pure Chemical, Osaka, Japan; doxorubicin, catalase (EC1.11.1.6, from bovine liver, 41,000 unit/mg protein) from Sigma-Aldrich Inc., St. Louis, MO, USA; mitomycin C from Merck KGaA, Darmstadt, Germany; 5-fluorouracil (5-FU) from Kyowa, Tokyo, Japan; methotrexate from Nacalai Tesque, Inc., Kyoto, Japan; docetaxel from Toronto Research Chemicals, New York, NY, USA; gefitinib from LC Laboratories^®, PKC Pharmaceuticals, Inc., Woburn, MA, USA; SBA (MW = 286) from ChemiScience, Tokyo, Japan; culture plastic dishes and plates (96-well) from Becton Dickinson Labware, Franklin Lakes, NJ, USA. eugenol was dissolved in DMSO at 400 mM before use, and diluted with medium. As a control, cells treated with 0.5% DMSO were used.

Reagents included De Man Rogosa and Sharpe (MRS) media (Becton Dickinson Difco, U.S.A.), hipolypeptone (Nihon Seiyaku, Japan), beef extract (MP Biomedicals, LLC, France), yeast extract (Becton Dickinson Difco), TBO (Waldeck, Munster), and Percoll (GE Healthcare Life Sciences, Japan). Glucose, Tween 80, K₂HPO₄, sodium ascorbate, L-cysteine-HCl, NaNO₃, MgSO₄, KH₂PO₄, NaH₂PO₄, NaCl, and 4′,6-diamidino-2-phenylindole (DAPI) were procured from Fujifilm Wako Pure Chemical Corporation, Japan. Data were collected using a spectrophotometer (UV-1200, Shimadzu, Japan) and a fluorescence microscope DMRXA/RD (Leica Microsystems, Germany). Cultures were prepared using fixed-type (model of rotor: BN 4–6) (H-201FR, Kokusan, Japan) and swing-type (model of rotor: RF 110) centrifuges (H-500FR, Kokusan). Deionised and doubly distilled water was used in all experiments.