Ultrapure hno3

“Wild-type” BY4741 yeast cells were pregrown in SRaf medium then shifted to MM/Gal medium and grown to density 5 × 10⁶ cells/mL before Ag(I) was added to the desired concentration. Similarly, cells transformed with recombinant MT cDNAs and the myrGFP control vector were pregrown in SRaf-Ura then shifted to MM/Gal-Ura for transgene induction and grown to density 5 × 10⁶ cells/mL before Ag(I) was added to the desired concentration. Ag(I) was added from a sterile 0.1 M AgNO₃ stock. To measure the metal accumulated, cells were harvested by centrifugation (1 min, 5000 rpm, 4 °C) and washed three times with ice-cold 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES)-Tris buffer containing 10 mM KCl, pH 6.0, before being suspended in deionized water (final density, 10⁸ cells/mL). For metal assay, cells were digested with 65% ultrapure HNO₃ (Merck, Darmstadt, Germany). Metal analysis was done using an instrument with a single collector, quadrupole inductively coupled plasma with mass spectrometry (ICP–MS, Perkin-Elmer ELAN DRC-e, Concord, ON, Canada) against Multielement ICP Calibration Standard 3, matrix 5% HNO₃ (Perkin Elmer Pure Plus). The metal cellular content was normalized to total cellular proteins, assayed spectrophotometrically [23 (link)]. Values were expressed as the mean ± standard error of the mean (SEM) of triplicate determinations on three independent yeast transformants.

Metal content of yeast cells was assessed as described in [54 (link)], with slight modifications. Cells exponentially growing on YPD were washed and suspended in MM liquid medium to OD₆₀₀ = 0.1 in the absence or presence of individual polyphenols (100 μM, final concentration). The cells were incubated with shaking (200 rpm) for 16 h at 30 °C before they were harvested and washed three times with ice-cold 10 mM 2-(N-morpholino)ethanesulfonic acid (MES)–Tris buffer, pH 6.8. Cells were finally suspended in deionized water (10⁸ cells/mL) and used for both metal and cell protein assays. Metal content analysis was done using an instrument with a single collector, quadrupole inductively coupled plasma with mass spectrometry (ICP-MS, Perkin-Elmer ELAN DRC-e, Concord, Vaughan, ON, Canada) with axial field technology for trace elements, rare earth elements, and isotopic analyses. Metal analyses were performed after digestion of cells with 65% ultrapure HNO₃ (Merck, Darmstadt, Germany). Standard solutions were prepared by diluting a 10 µg/mL multielement solution (Multielement ICP Calibration Standard 3, matrix 5% HNO₃, PerkinElmer Pure Plus, Shelton, CT, USA). The cellular metal content was normalized to total cellular proteins, which were assayed spectrophotometrically [55 (link)].

Every specimen was thermally treated at 600 °C for 3 h in an electric furnace, and weighed before and after the thermal treatment. In order to fabricate an inorganic solution from the specimen residue, the remaining residue was transferred to a Teflon vessel containing 1 M ultrapure HNO₃ (Sigma-Aldrich, Saint Louis, MI, USA). The vessel was placed in a microwave reactor for 60 min. In microwave-stimulated hydrothermal conditions, under autogenous pressure of 20 atm and at 250 °C, the solution was obtained. The procedure was repeated for every specimen. Resulting solutions were analyzed for the content of elements (Ca, Cd, Cr, Cu, Fe, Mg, Mn, Pb, Zn) using inductively coupled plasma optical emission spectrometry (ICP-OES). The total concentration of the trace elements was measured using an Agilent 5110 synchronous vertical dual view (SVDV) ICP-OES instrument equipped with an easy-fit quartz torch with standard 1.8 mm injector and a Seaspray nebulizer as a sample introduction system and a double-pass glass cyclonic spray chamber. The resulting solutions were measured versus simple standard solutions. The instrument was run under standard operating conditions, while combining radial and axial acquisition of radiation emitted by the vertical plasma over the entire wavelength range, in a single measurement.

Strontium nitrate was obtained from Wako Pure Chemical Industries (Osaka, Japan). Sr standard solution (1 mg mL⁻¹) for atomic absorption spectrometry was also from Wako Pure Chemical Industries. Ultrapure HNO₃ was purchased from Sigma Aldrich (Tokyo, Japan). ⁹⁰SrCl₂ (1.05 × 10⁴ Bq mL⁻¹) in 0.1 N HCl was purchased from Japan Radiation Association (Tokyo, Japan).