DSS 2,2-dimethyl-2-silapentane-5-sulfonic

Fig. 5. Proton magnetic resonances of diamagnetic molecules. DSS (2,2-dimethyl-2-silapentane-5-sulfonate) is used as an internal reference. On a HR-220 spectrometer 1 ppm (part per million) corresponds to 220 cps (cycles/sec.)...

DSS 2,2-dimethyl-2-silapentane-5-sulfonate (usually as the sodium salt)... 

The proton chemical shift of H20 is 4.73 ppm downfield from that of the methyl group of 2,2-dimethyl-2-silapentane 5-sulfonate (DSS) at 29°C. [Modified from Ho and Russu (1981)]. The particular resonance can be observed in both H20 and D20, unless specifically stated otherwise. 

This material has several advantages it is chemically inert, symmetrical, volatile (bp 27°C), and soluble in most organic solvents it gives a single, intense, sharp, absorption peak, and its protons are more shielded than almost all organic protons. When water or deuterium oxide is the solvent, TMS can be used as an external reference in a concentric capillary or the methyl protons of the water-soluble sodium 2, 2-dimethyl-2-silapentane-5-sulfonate (DSS), (CH3)3SiCH2CH2CH2S03Na, are used as an internal reference (0.015 ppm). 

DSS is 3-(trimethylsilyl)-l-propane sulfonic acid, sodium salt (or sodium 2,2-dimethyl-2-silapentane-5-sulfonate). TSP is sodium -3-trimethylpropionate -2, 2, 3, 3-d. Both are reference standards used in aqueous solutions. 

TMS cannot be used as an internal reference for substances dissolved in DjO. For aqueous solutions, the standard used is often the sodium salt of 2,2-dimethyl-2-silapentane-5-sulfonic acid or DSS. 

All proton NMR spectra were recorded on a Varian Unity 600 at 25 C. 6 to 10 mg of the disulfide linked c-Myc-Max heterodimeric LZ were dissolved in 0.5 mL of potassium phosphate buffer (50 mM, 10% DiO / 90% H2O and pH 4.7) containing 100 mM KCl and ImM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) to yield solutions ranging from 0.75 to 1.25 mM. Proton resonances were assigned from two-dimensional double quantum filtered correlation spectroscopy (DQF-COSY (21)), two-dimensional total correlation spectrocopy (TOCSY mixing time = 50 ms (22)) and two-dimensional nuclear Overhauser enhancement spectrocopy (NOESY mixing times = 150 and 200 ms (23)) experiments. Sequential assignment of the proton resonances was performed as described by Wuthrich (24). 

The H nmr spectrum in D20 exhibits a single resonance at 2.52 ppm for the methyl protons with DSS, the sodium salt of 2,2-dimethyl-2-silapentane-5-sulfonic acid as reference. The electronic spectrum for an aqueous solution is characterized by an absorption maximum at 358 nm (e = 7.4 Lrnole 1 cm 1) in H20. Other physical properties are described in the literature [the checkers report 5(CH3) = 2.6 ppm with Xmax = 336 nm, e = 650 Lmol 1 cm"1 ]. 

Nuclear Magnetic Resonance (NMR) Spectrometry. Spectral verification of derivatization was provided by proton NMR of N-acetylated and carboxylic- and hydroxyl-derivatized heparins using a 300-MHz Varian SC 300 NMR spectrometer. Forty milligrams/milliliter of the respective heparin was dissolved in deuterium oxide with 1% 2,2-dimethyl-2-silapentane-5-sulfonate(DSS) and transferred to 5-mm NMR tubes. Spectra were obtained at room temperature with a spin rate of 20 rps and a gas flow rate of 13 ft3/h. 

For successful NMR experiments a protein solution of high purity (>95%), stability (over a week), and appropriate concentration (0.1-1 mM) is needed the total sample volume and mass should be 350-550 uL and 3-30 mg, respectively. 2,2-Dimethyl-2-silapentane-5-sulfonic acid (DSS) rather than tetramethylsUane (TMS) is used as a reference compound. Although both natural and synthetic polypeptides and proteins can be used, most samples are expressed via a suitable prokaryotic or eukaryotic cellular or ceU-free recombinant technique. The latter method eliminates most toxic effects attributed to the overproduction of recombinant proteins. Biotechnological approaches have the common advantage of large-scale protein production, specific or non-specific stable isotope labeling, and easy sequence variation. 

The liquid NMR spectrum of the M-DME used was obtained on a Bruker Avance 400 FT NMR spectrometer. Chemical shifts were calculated relative to DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfonate) dissolved in D2O. The spectrum was recorded at 100 Ml 1/ for approximately 3000 transients. All the spectra were recorded with a relaxation delay of 2 s and the chemical shifts were accurate to 1 ppm. 

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