NMR spectroscopy experiments were carried out in Bruker Avance 700 MHz with triple resonance z-axis gradient cryoprobe and Bruker DRX 600-MHz spectrometer equipped with a cryoprobe at 298 K. Uniformly 15N labeled L-PGDS sample was prepared in 50 mM sodium phosphate buffer, pH 7.5 at a concentration of 0.25 mM with 5% D2O and 50 μM DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid, used as NMR standard for calibration). To identify the key residues of L-PGDS involved in heme binding, two dimensional 1H–15N HSQC spectrum of 15N labeled WT refolded L-PGDS was recorded at 25°C as a reference. Freshly prepared heme from a 2.5 mM stock solution was prepared in 0.1% NaOH and was gradually added into the L-PGDS sample before acquiring the 2D 1H–15N HSQC spectrum at each titration step. Spectra were referenced with respect to DSS and then overlapped to check if the cross-peaks showed any shift or change in signal intensity. Data were processed in Topspin 3.5 (Bruker Corporation) and then analyzed using computer-aided resonance assignment (CARA) (www.nmr.ch ) [47 ].
Drx 600 mhz
Manufactured by Bruker
Sourced in Germany
The DRX 600 MHz is a high-performance nuclear magnetic resonance (NMR) spectrometer designed for advanced analytical applications. It offers a magnetic field strength of 600 MHz, providing high-resolution data for the structural elucidation and identification of chemical compounds.
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4 protocols using drx 600 mhz
1
Probing Heme Binding Interactions in L-PGDS
2
NMR Spectroscopy of Deuterated Samples
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All NMR 1H-13C dept-HSQC spectra were recorded on a Bruker DRX 600 MHz instrument. About 3 mg of each sample were dissolved in 0.5 mL of 99.9% deuterium oxide. All spectra were recorded at 298 K with HOD suppression by pre-saturation. 1H-13C dept-HSQC spectra were run with 1024 × 256 points and globally optimized alternating phase rectangular pulses for decoupling. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm for 1H and to methanol for 13C.
3
Spectroscopic Analysis of Organic Compounds
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1H-NMR and 13C-NMR spectra were recorded on Bruker DRX 600 MHz and Bruker Avance III 400 MHz (Bruker Daltonics, Billerica, MA). ESI-MS spectra were recorded by “Waters” 3100 “USA", TQ Detector (Acquity ultra performance LC), Mass lynx V 4.1. Chemical shifts are given in values (ppm) relative to trimethylsilane as an internal reference. Silica gel 60, particle size 0.063–0.2mm, 70–230 mesh (Merck, Darmstadt, Germany) was used for column chromatography. For thin layer chromatography (TLC) aluminum sheet Silica gel 60 F254 pre-coated plates (Merck, Darmstadt, Germany) were used.
4
NMR Spectroscopy Protocol for Compound Analysis
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1H NMR spectra were acquired using a Bruker DRX 600 MHz spectrometer (Bruker Biospin, Karlsruhe, Germany), operating at 300 K, using the standard 1‐dimensional NMR pulse sequence [relaxation delay‐90°‐t1‐90°‐tm‐90°‐acquire‐FID]. The water peak was suppressed by selective irradiation during the relaxation delay of 2 s and mixing time (tm) of 100 ms; t1 was fixed to 2 μs. The 90° pulse length was adjusted to approximately 10 μs. A total of eight dummy scans and 64 scans were recorded into 32 k data points with a spectral width of 20 ppm. An exponential function corresponding to a line broadening of 0.3 Hz was applied to each free induction decay (FID) prior to Fourier transformation15 .
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1H NMR spectra were acquired using a Bruker DRX 600 MHz spectrometer (Bruker Biospin, Karlsruhe, Germany), operating at 300 K, using the standard 1‐dimensional NMR pulse sequence [relaxation delay‐90°‐t1‐90°‐tm‐90°‐acquire‐FID]. The water peak was suppressed by selective irradiation during the relaxation delay of 2 s and mixing time (tm) of 100 ms; t1 was fixed to 2 μs. The 90° pulse length was adjusted to approximately 10 μs. A total of eight dummy scans and 64 scans were recorded into 32 k data points with a spectral width of 20 ppm. An exponential function corresponding to a line broadening of 0.3 Hz was applied to each free induction decay (FID) prior to Fourier transformation
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