Pda md 2018 detector

Size-exclusion chromatography was performed by analytical HPLC (Jasco LC-2000 Plus) equipped with a PU 2880 Plus injector and a PDA MD 2018 detector (Jasco). The samples (50 μM in 100 mM Tris-HCl at pH 7.4) were separated by a system containing a Phenomenex BioSep-SEC-S3000 column (7.8 × 300 mm, 5 μm, resolution range of 15 to 2000 kDa, Phenomenex, Inc., Torrance, California, USA) using a flow rate of 1.0 mL/min in 100 mM Tris-HCl buffer (pH 7.4) and 50 mM NaCl. The elution profile was monitored by absorbance (λ = 280 nm). Bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B₁₂ (1.35 kDa) were used as molecular weight standards (Bio-Rad). The chromatograms were analyzed using Jasco BORWIN, version 1.50, software (Jasco). The enzymes were reduced or oxidized by treatment with 5 mM TCEP or 1.2 molar excess of H₂O₂, respectively, for 30 min at 25 °C.

Size-exclusion chromatography experiments were performed by analytical HPLC equipped with a PU 2880 Plus injector and a PDA MD 2018 detector (LC-2000 series; Jasco, Tokyo, Japan). The samples (50 μM in 100 mM Tris-HCl, pH 7.4) were separated by a system containing a Phenomenex BioSep-SEC-S3000 column (7.8 × 300 mm, 5 μm, resolution range of 1–300 kDa, Phenomenex, Inc., Torrance, California, USA) at a flow rate of 1.0 mL min⁻¹ in 100 mM Tris-HCl, pH 7.4, containing 50 mM NaCl. The elution profile was monitored by absorbance at λ = 280 nm. Bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa) were used as molecular standards (Bio-Rad Laboratories, Richmond, USA). Chromatograms were analyzed using Jasco BORWIN, version 1.50, software (Jasco, Tokyo, Japan). The redox treatments for rPbPrx1 were either 5 mM TCEP (reductant) or 1.2 molar equivalent of hydrogen peroxide (oxidant) for 30 min, at 25°C prior to the chromatographic runs.