Lambda 20

Time courses of Dbp5 (0.6–1 μM) steady-state ATPase assayed by absorbance change at 340 nm using the NADH-coupled assay were acquired on a Perkin-Elmer Lambda 20 UV–Vis spectrophotometer thermostatted at 25 °C [13] (link), [43] (link), [66] . The [ATP] dependence of the initial steady-state ATPase rate (v₀) was fitted to the Michaelis–Menten equation: $v_0=\frac{k_{\mathrm{cat}}{\left[\text{H}\right]}_{\text{T}}\left[\text{T}\right]}{\left[\text{T}\right]+K_{\text{M,ATP}}}$ where k_cat is the maximal turnover rate of Dbp5 at saturating ATP in units of s^− 1, [H]_T is the total Dbp5 concentration, K_M,ATP is the Michaelis constant for ATP, and [T] is the total [ATP]. The [ADP] under our conditions of 2 mM ATP is ∼ 7 μM [43] (link). Steady-state ATPase assays were also performed using ATPγS as a substrate.
RNA-stimulated steady-state ATPase experiments were performed as detailed above, using 100 nM Dbp5 and 20 mM ATP. Data were fitted to the quadratic form of the Briggs–Haldane equation $v_{\mathrm{o}\mathrm{b}\mathrm{s}}=\left(k_{\mathrm{cat}}-k_0\right)\frac{{\left[\mathrm{H}\right]}_{\mathrm{T}}+{\left[\mathrm{R}\right]}_{\mathrm{T}}+K_{\mathrm{M},\mathrm{RNA}}-\sqrt{{\left({\left[\mathrm{H}\right]}_{\mathrm{T}}+{\left[\mathrm{R}\right]}_{\mathrm{T}}+K_{\mathrm{M},\mathrm{R}\mathrm{N}\mathrm{A}}\right)}^2-4{\left[\mathrm{H}\right]}_{\mathrm{T}}{\left[\mathrm{R}\right]}_{\mathrm{T}}}}{2{\left[\mathrm{H}\right]}_{\mathrm{T}}}+k_0$ where [R]_T is the total RNA concentration in nucleotides [14] (link), [66] .

The cell growth was determined periodically by measurement of the optical density (OD₆₀₀) of the cultures at 600 nm using a UV spectrophotometer (Lambda 20, Perkin Elmer, USA). The gases present in the headspace of the serum bottles were measured by gas chromatography (DS6200 Donam Systems Inc., Seoul, Korea) fitted with a column and thermal conductivity detector. The amounts of glucose, ethanol, and other metabolites were quantified using high-performance liquid chromatography (Agilent Technologies, HP, 1200 series) installed with an Aminex carbohydrate analysis column, as described in Sankaranarayanan et al. [40 (link)]. Protein-expression analysis was performed by SDS-PAGE as described earlier [41 (link)]. The protein present in the samples used for determination of enzymatic activity was measured by Bradford assay as described previously [42 (link)].

The UV-Vis spectrophotometer (Lambda 20, Perkin Elmer, Buckinghamshire, UK) with 1 cm matched quartz cells was used for all absorbance measurements in the range from 200 to 400 nm.
The Atlas Suntest CPS + (Atlas, Mount Prospect, IL, USA) and the thermostat ST-1 + (Pol-Eko, Wodzisław Śląski, Poland) were used for the exposure of substances in highly accelerated and stress testing. The irradiation chamber was equipped with a temperature control system (35–100 °C), 1500 W air-cooled xenon lamps, and direct setting and control of irradiance in a wavelength range of 300–800 nm. A photostability study of solutions of the tested substances was performed in quartz cuvettes with a stopper, 10 mm, 3 mL (Merck, Darmstadt, Germany).
Stress tests were performed in a Thermal Testing Chamber KBC-100 (Wamed, Warsaw, Poland) with an accuracy of heat set temperature of ±1.0 °C.
The solutions were sterilized using a small water steam sterilizer (Extacta, M.O. Com S.R.L., Montecchio Emilia, Italy).

The cell concentration was determined in a 10-mm-path-length cuvette using a double-beam spectrophotometer (Lambda 20, Perkin-Elmer, Norwalk, CT). The concentrations of 3-HP were determined by HPLC using a slightly modified version of the method described elsewhere [29 (link)]. Briefly, the obtained by 10 min centrifugation of the culture samples at 10,000×g was filtered through a Tuffryn-membrane (Acrodisc; Pall Life Sciences, Port Washington, NY) and eluted through a 300 mm × 7.8 mm Aminex HPX-87H (Bio-Rad, USA) column at 65 °C using 2.5 mmol/L H₂SO₄ as the mobile phase.

Nordihydroguaiaretic acid (NDGA), Trolox, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), Soybean LOX linoleic acid sodium salt were purchased from the Aldrich Chemical Co. Milwaukee, WI, (USA). Phosphate buffer (0.1 M and pH 7.4) was prepared mixing an aqueous KH₂PO₄ solution (50 mL, 0.2 M), and an aqueous of NaOH solution (78 mL, 0.1 M); the pH (7.4) was adjusted by adding a solution of KH₂PO₄ or NaOH). For the in vitro tests a Lambda 20 (Perkin–Elmer-PharmaSpec 1700) UV–Vis double beam spectrophotometer was used.

UV-Vis measurements were performed on a Perkin-Elmer Spectrophotometer. Absorption, utilising Starna Silica (quartz) cuvette with 10 mm path lengths, two faces polished. Data was collected via the Perkin-Elmer Lambda 20 software package. Further reprocess of the data was operated in OriginPro 8.0 graphing software.