Avance drx 600

Samples were resuspended in 0.6 ml NMR buffer consisting of 0.1 M phosphate buffer pH 7.4, 1 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP), and 0.55 ml was transferred to a 5 mm NMR tube. Spectra were acquired on a Bruker Avance DRX600 NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany), with 1H frequency of 600 MHz, and a 5 mm inverse probe. Samples were introduced with an automatic sampler and spectra were acquired following the procedure described by Beckonert et al [15 (link)]. Briefly, a one- dimensional NOESY sequence was used for water suppression; data were acquired into 64 K data points over a spectral width of 12 KHz, with 8 dummy scans and 512 scans per sample. In addition, 2D ¹H-¹H COSY were acquired. Data were acquired into 512 × 4096 data points covering 6 × 6 KHz, with 3 scans for each increment.
Spectra were processed in iNMR 2.6.3 (Nucleomatica, Molfetta, Italy). Fourier transform of the free-induction decay was applied with a line broadening of 0.5 Hz. Spectra were manually phased and automated first order baseline correction was applied. Metabolite assignments were based on Tredwell et al [14 (link)]. Metabolite concentrations from 2D spectra, relative to the internal standard TSP were calculated using rNMR [16 (link)], and data were normalised by the probabilistic quotient normalisation method described by Dieterle et al [17 (link)].

All ¹H NMR spectra were acquired at 300 K on a Bruker Avance DRX-600, operating at 600.13 MHz (Bruker Biospin, Wissembourg, Germany), equipped with an autosampler and an inverse ¹H-¹³C-¹⁵N cryoprobe. For urine samples, ¹H NMR spectra were acquired using a standard pulse sequence NOESY to suppress water resonance. A relaxation delay of 2 s and mixing time of 150 ms were used and 256 free induction decays (FIDs) were collected into 32 k data points using a spectral width of 20 ppm with an acquisition time of 1.36 s. ¹H NMR spectra were acquired using the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence with pre-saturation, with a total spin-echo delay (2 nt) of 240 ms to attenuate broad signals from proteins and lipoproteins. A total of 128 transients were collected into 32 k data points using a spectral width of 20 ppm, a relaxation delay of 2 s and an acquisition time of 1.36 s.

For NMR measurements, 500 μM of GL-bound or heme-dimerized PGRMC1 were prepared in 50 mM phosphate buffer (pH 7.0) containing 5% D₂O. NMR spectra were measured at 25 °C using a Bruker Avance DRX 600 spectrometer equipped with a triple resonance (¹H/¹³C/¹⁵N) cryogenic probe. Transverse relaxation-optimized spectroscopy (TROSY) types of 2D ¹H-¹⁵N heteronuclear single-quantum coherence (HSQC) spectra were measured. NMR data were processed using the program NMRPipe [36 (link)], and signal assignments were performed using the programs Kujira [37 (link)], and NMRView [38 (link)]. A docking model structure of the GL-bound PGRMC1 was calculated using the program HADDOCK 2.1 [39 (link)] using the crystal structure of PGRMC1 (PDB: 4X8Y) and the chemical shift data, and residues showing Δδ larger than 0.05 ppm were defined as the GL-binding site.

NMR spectra were recorded on a Bruker Avance DRX 600 equipped with a cryoprobe operating at 600 MHz for proton in D₂O solution containing 1 mM sodium 3-trimethylsilyl [2,2,3,3-D₄] propionate as a chemical shift reference for ¹H spectra. Water was deionized and purified (Milli-Q, Millipore, Germany), whereas TRI Reagent^® and all solvents were purchased from Sigma (Aldrich, Milan, Italy). Chromabond^® HR-X resin was obtained from Macherey-Nagel GmbH (Düren, Germany). Ultrafiltration was carried out by Vivaspin 500 centrifugal concentrators (Sigma-Aldrich) with 3, 10 and 30 kDa molecular weight cut-off membrane of polyethersulfone (PES). Electrophoresis (SDS-PAGE) was performed by a mini gel apparatus BioRAD (Milan, Italy). To estimate the molecular mass, Precision Plus Protein™ Dual Color Standards (BioRad, Hercules, CA, USA) with 10 recombinant proteins of precise molecular weights (10–250 kD) were used.

Optical rotations were measured on a Jasco P2000 digital polarimeter (Jasco, Cremella, Italy). UV spectra were acquired on a Jasco V-650 spectrophotometer, NMR spectra were recorded on a Bruker Avance DRX 600 (Bruker, Milan, Italy) equipped with a cryoprobe operating at 600 MHz for protons. Chemical shift values are reported in ppm (δ) and referenced to internal signals of residual protons (CD₃OD ¹H δ3.34, ¹³C 49.0 ppm). High-resolution mass spectra were acquired on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Milan, Italy); HPLC analyses have been performed on a Jasco system (PU-2089 Plus-quaternary gradient pump equipped with a Jasco MD-2018 Plus photodiode array detector). [1-¹³C]-acetate, [2-¹³C]-acetate and [1,2-¹³C₂]-acetate, [1-¹³C]-glycolate (all sodium salts), and salicylhydroxamic acid (SHAM) were obtained from Sigma Aldrich (Milan, Italy). Solvents were purchased from VWR (Milan, Italy) and were HPLC-grade.