Microcon ultracel ym 50 ultrafiltration devices

NADPH was purchased from AppliChem (Darmstadt, Germany). Dithionitrobenzoic acid (DTNB), sodium selenite and DEAE resin were all obtained from Sigma-Aldrich (St. Louis, MO). Phenyl sepharose resin was from Pharmacia-Amersham Biosciences (Uppsala, Sweden). Microcon Ultracel YM-50 ultra-filtration devices Millipore (Billerica, MA) were used for concentrating enzyme samples. Resin for peptide synthesis (2-chlorotritylchloride) was from Novabiochem (San Diego, CA). Fmoc amino acids were from Synbiosci Corporation (Livermore, CA), except for Fmoc-homocysteine, which was from Bachem (King of Prussia, PA). Primers for mTR3 mutants were from IDT (Coralville, IA). Plasmid pTYB3 and restriction enzymes were from New England Biolabs (Ipswich, MA). The production and purification of the recombinant and semisynthetic enzymes used in this study have been previously reported (6 (link), 19 (link), 24 (link), 25 (link), 30 (link)). The selenium content of the wild type (WT) semisynthetic enzyme is 91% as reported in (19 (link)). Enzyme kinetic assays were performed on a Cary50 UV-Vis spectrophotometer (Walnut Creek, CA) and all enzymatic assays were conducted at room temperature unless otherwise noted. All other chemicals were from Fisher Scientific or Acros Organics (Morris Plains, New Jersey). Aryl disulfides were prepared by Watson Lees and others as described in (31 (link)–35 (link)).

NADPH was purchased from AppliChem (Darmstadt, Germany). DEAE resin was obtained from Sigma-Aldrich (St. Louis, MO). Phenyl sepharose resin was from Pharmacia-Amersham Biosciences (Uppsala, Sweden). Microcon Ultracel YM-50 ultrafiltration devices by Millipore (Billerica, MA) were used for concentrating enzyme samples. 2-chlorotritylchloride resin was from Novabiochem (San Diego, CA). Fmoc amino acids were from Synbiosci Corporation (Livermore, CA), except for Fmoc-homocysteine, which was from Bachem (King of Prussia, PA). CLEAR-OX resin was from Peptides International (Louisville, KY). Primers for DmTR mutants were from IDT (Coralville, IA), plasmid pTYB3, and restriction enzymes were from New England Biolabs (Ipswich, MA). The DmTR-SG construct was synthesized by Retrogen, Inc. (San Diego, CA). Enzyme kinetic assays were performed on a Cary50 UV-Vis spectrophotometer (Walnut Creek, CA) and all enzymatic assays were conducted at room temperature unless otherwise noted. All other chemical were from Fisher Scientific (Fair lawn, NJ) or Acros Organics (Morris Plains, New Jersey).