Pd minitrap g 25

GTP hydrolyzing activity was measured under different buffer conditions, as reported in the Results section, using the SensoLyte® MG Phosphate Assay Kit (AnaSpec), based on the colorimetric reaction involving malachite green reagent, molybdate and orthophosphate, as previously reported for other UreG proteins^{22 (link)}. Before the analysis, the protein buffer was exchanged using PD MiniTrap G-25 desalting columns (GE Healthcare) according the manufacture’s instructions. The reaction mixture, containing 20 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 5 mM MgCl2, 0.4 mM GTP, 1 mM DTT and 10 µM SpUreG, in the absence or in the presence of 1 or 3 M GuHCl, 2 mM SDS, 1 or 2 M TMAO and 40% TFE, was incubated at 37 °C, at 50 °C and at 70 °C. Aliquots (100 µl) were removed at different incubation times and diluted with 400 µL of H₂O and 100 µL of malachite green solution. Phosphate concentration was determined measuring the absorbance of the solution after 20 minutes of incubation, according to a calibration curve performed using phosphate standard solutions. Each experiment was conducted in triplicate. Phosphate concentrations measured for blank experiments, performed in parallel under the same experimental conditions, were subtracted and the resulting data were used to derive the turnover number.

VLPs were stained with DiD (Thermo Fisher Scientific, Eugene, OR) at 20 µg/mL for 30 min at 22 °C, and then purified from free dye using gel filtration columns (PD MiniTrap G-25, GE Healthcare, Buckinghamshire, UK). MDMs were detached from plastic plate surface using Accutase Cell Detachment Solution (BioLegend, San Diego, CA) and plated on 96-well Nunclon Delta black flat-bottom plates (Thermo Fisher Scientific, Roskilde, Denmark) at 5 × 10⁴ cells/well. The following day, MDMs were exposed to DiD-labeled VLPs (HA concentration 15.0 µg/mL) at 4 °C for 1 h. Endocytosis inhibitors were applied in ice-cold medium supplemented with 10% FBS: dynasore hydrate 50 µM, genistein 200 µM, amiloride hydrochloride 1 mM, cytochalasin D 4 µM (all from Sigma-Aldrich, St. Louis, MO). DiD fluorescence was measured with pre-heated (37 °C) spectrophotometer (Infinite 200 PRO, Tecan, Männedorf, Switzerland) at 15-min intervals over 2 h. Fusion efficiency was determined following the addition of Triton X-100 (Sigma-Aldrich) to each well (final concentration 1%) to obtain full DiD dequenching.

Protein solutions were diluted to a concentration of 0.3 mg/ml in a buffer containing 5 mM KP_i pH 7.5, 15 mM KCl. Denaturation of β-cardiac myosin was measured by monitoring the temperature-dependent changes of ellipticity at 222 nm in a temperature controlled π*-180 Spectrometer equipped with a circular dichroism unit (Applied Photophysics, Leatherhead, UK). Measurements were performed in a 0.3-cm path length Quartz cuvette with a temperature gradient of 1°C min⁻¹. The transition midpoint of the unfolding reaction was calculated by fitting a Boltzmann function to the experimental data. Prior to measurements, the storage buffer of β-cardiac myosin that contains DTT and sucrose were exchanged using PD MiniTrap G-25 (GE Healthcare, Little Chalfont, UK) size exclusion columns equilibrated with CD Buffer (5 mM KP_i pH 7.5, 15 mM KCl).

Fluorescein isothiocyanate isomer I (FITC) was dissolved in DMSO to 80 μM, and then added to HFn or Gd-HFn solution (4 μM in the buffer of 100 mM carbonate pH 9.0). The mixture was incubated at 4 ℃ overnight, and then purified with a PD MiniTrap G25 column (GE Healthcare) to remove the free FITC. The concentration of conjugated FITC was determined by measuring its optical density at 492 nm, the concentration of the HFn protein was determined using a BCA Protein Assay Kit.

LDL (d=1.006–1.063 g/ml) and HDL (d=1.063–1.210 g/ml) were isolated by ultracentrifugation as described previously [18 (link)]. Isolated LDL and HDL from healthy volunteers were dialysed against PBS. HDL from patients was applied to PD-MiniTrap G-25 (GE Healthcare) chromatography columns to eliminate potassium bromide (KBr). Lipoproteins were stored at −80 or 4°C until used for the antioxidant ability assay or other experiments, respectively.