Dd2 700 mhz spectrometer

The NMR spectra were recorded and measured using an Agilent DD2 700 MHz spectrometer (Santa Clara, CA, United States). Deuterated dimethylsulfoxide (DMSO-d6, Sigma-Aldrich) was used as NMR solvent and tetramethylsilane (Sigma-Aldrich) was used as an internal standard. Samples were placed in 5 mm NMR sample tubes (Sigma-Aldrich) and the analysis was performed at an elevated temperature of 25°C to improve spectral resolution.

The experiments were performed on a 600 MHz Varian VNMRS spectrometer using a room temperature XH broadband probe (X-nucleus on the inner coil). Additional ²³Na NMR measurements were performed on an Agilent DD2 700-MHz spectrometer using OneNMR direct detect broadband probe. ²³Na⁺ NMR experiments were performed in the presence of either 50 μM or 25 μM A_2AR. An external ²³Na reference was prepared by mixing 50 mM NaCl and 50 mM TmCl₃ in equal volumes prior to loading into the coaxial insert. The resultant ²³Na reference was conveniently shifted downfield by 4 ppm from the free sodium chloride signal associated with the receptor sample. A typical experimental setup included a 7.5 μs 90° excitation pulse, an acquisition time of 250 ms, a spectral width of 15 kHz, and a repetition time of 1.25 s. Most spectra were acquired with 512 scans and yielded a signal to noise ratio greater than 100. Information regarding binding can be reliably obtained by observing the shift of the free peak and line broadening associated with fast exchange between “free” and “bound” states.

¹H-NMR was performed to monitor the activity of FjoAcXE and AxyAgu115A on Ac-XOS substrates. Reactions comprised 1% (w/v) acetylated (glucurono)-xylooligosaccharides in 50 mM HEPES buffer (pH 7.0) and 10 µg of each protein; the final reaction volume was 400 µL. The reaction continued for 20 h at 30 °C and gentle shaking. Reaction mixtures without enzyme were used as negative controls. Following the incubation, samples were filtered through Acrodisc^® syringe filters with 0.2-µm Supor^® membrane (Pall Corporation), and lyophilized. The samples were then dissolved in 300 µL D₂O and transferred into 3-mm NMR tubes (Norell) for analysis using an Agilent DD2 700 MHz spectrometer equipped with a triple resonance HCN cold probe with a scan number of 64, relaxation delay of 1 s and acquisition time of 4.5 s. The data were obtained using VnmrJ 4.0 (Agilent) and analyzed with MestReNova 10.0 (Mestrelab Research). The HDO peak at 4.790 was used as internal standard. The change in signal intensity in the regions between 5.4 and 4.4 ppm corresponding to acetylated Xylp residues in the anomeric region of the spectrum, and 2.30–2.05 ppm region corresponding to the acetyl group methyl protons were used to assign proton chemical shifts, as reported in Uhliariková et al. [24 (link)].