Guanidinium hydrochloride

Gel filtration separations are performed in an aqueous environment and the requirement on chemical resistance is therefore modest. Most column tubes and accessories (e.g., support net, sealings) withstand the solvent mixtures sometimes used in aqueous SEC such as 20% ethanol, 3 M guanidinium hydrochloride, 6 M urea, or 0.1 M HCl and 1 M NaOH, the last two being used for cleaning the packed column (see Section V). 

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

If the protein of interest is a heteromultimer (composed of more than one type of polypeptide chain), then the protein must be dissociated and its component polypeptide subunits must be separated from one another and sequenced individually. Subunit associations in multimeric proteins are typically maintained solely by noncovalent forces, and therefore most multimeric proteins can usually be dissociated by exposure to pEI extremes, 8 M urea, 6 M guanidinium hydrochloride, or high salt concentrations. (All of these treatments disrupt polar interactions such as hydrogen bonds both within the protein molecule and between the protein and the aqueous solvent.) Once dissociated, the individual polypeptides can be isolated from one another on the basis of differences in size and/or charge. Occasionally, heteromultimers are linked together by interchain S—S bridges. In such instances, these cross-links must be cleaved prior to dissociation and isolation of the individual chains. The methods described under step 2 are applicable for this purpose. 

Nave, R., Weber, K., and Potschka, M., Universal calibration of size-exclusion chromatography for proteins in guanidinium hydrochloride including the high-molecular-mass proteins titin and nebulin,. Chromatogr. A, 654, 229, 1993. 

The presence of calcium in horseradish peroxidase was demonstrated originally by Haschke and Friedhoff, working with the C and A (imspec-ified, but likely to have been predominantly A2) isoenzymes (209). HRP C and HRP A contain 2.0 0.13 and 1.4 0.19 moles calcium per mole enzyme, respectively, as determined by atomic absorption spectroscopy. Incubation in 6 M guanidinium hydrochloride and 10 mM EDTA for 4 hours at neutral pH and room temperature gave calcium-depleted enzymes with specific activities decreased by 40% and 15%, respectively. The thermal stability of calcium-depleted HRP C was also reduced compared to native enzyme. Reconstitution was successful only with calcium-depleted HRP C (209). It remains to be established whether this reflects true structural differences between the calcium binding sites of the two isoenz5unes, or is a consequence of the relatively harsh... 

Renaturation after application of urea, guanidinium hydrochloride, or alcohols... 

Substances destroying the water structure, fhe so-called chao-tropic substances such as urea, guanidinium hydrochlorid, or some organic solvents such as mefhanol, efhanol, or acetonitrile, suppress hydrophobic interactions. Ions also alter the water structure. The power of destruction of water structure is given by the Hofmeister series ... 

As described for lEC, elution is done by a stepwise or a continuous change of buffer composition. The mildest elution buffer is an aqueous buffer with low ionic strength, e.g., 20 mm Tris-HCl. If it is not successful, desorb with a chaotropic solvent, e.g., 2 M potassium rhodanide (thiocyanate), 2.5 M guanidinium hydrochloride, up to 7 M urea, or with increasing concentrations of methanol or acetonitrile. Especially the use of rhodanide or urea may be accompanied by a chemical modification of amino acid side chains, which disturbs amino acid analysis. 

For a non-denaturating preparation take Soln. B-D without guanidinium hydrochloride and urea, respectively, and 8-mercaptoethanol and DTT, respectively. 

Fig. 1. SDS-PAGE pattern of human IgG heavy chain eluted from PVDF membrane. Human IgG (10 pg) was resolved by SDS-PAGE and transferred to a PVDF membrane. The proteins on the membrane were stained with amido black in water, and the heavy chain was excised and eluted with guanidinium hydrochloride/lysophosphatidylcholine. After precipitation with absolute alcohol, the glycoprotein was subjected to analytical SDS-PAGE, and the gel was stained with Coomassie brilliant blue to ascertain its purity. Lanes 1 and 4 original commercial preparation of human IgG lanes 2 and 3 IgG heavy chain eluted from the PVDF membrane lane 5 mixture of molecular mass standards, from top to bottom phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 ldDa), ct-lactalbumin (14.4 kDa).

The unfolded state U is modeled as a single conformation even though this is by no means obvious and usually cannot be verified. The unfolding equilibrium between N and U can be shifted to the unfolded form by measures such as an increase in temperature or an increase in the concentration of structure breakers , termed chaotropes, such as urea or guanidinium hydrochloride. When either temperature or the concentration of chaotropes is decreased (or the concentration of structure formers , kosmotropes, is increased), the folding equilibrium reverts to the native state N. The two-state model is an approximation very often, analytically verified folding intermediates render the two-state model incomplete. 

In early work, Schmuck synthesised the simple 2-guanidiniocarbonylpyrrole 23 (Figure 21) by the reaction of ethyl-l/f-pyrrole-2-carboxylate with guanidinium hydrochloride in the presence of sodium methoxide affording the receptor in 52% yield.17... 

RNA from Animal Tissues and Cells Using Guanidinium Hydrochloride... 

Reaction buffer 0.5 M Tris-HCl, 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5 containing 6 M guanidinium hydrochloride. 

Scheme 2. Ellmans sulfonamide linker allowed the (DIPEA, N-ethyl-N,N-diisopropylamine DTT, synthesis of the acid- and base-labile glycopeptide dithiothreitol EDT, ethanedithiol GnHCI, thioester 6, which was used for the Native guanidinium hydrochloride NMP, N-methyl-...

With 500 pl/well of 8 M guanidinium hydrochloride solution, the bound stain is then extracted for 24 h at +4 °C. After shaking the plates gently for 10 min, 100 pl/well of each supernatant is then transferred to round-bottom microtiter-plates, and the extinction photometrically assessed in the plate-reader at a wavelength of 610 nm. 

The pellets are carefully drained and dissolved in 6 M guanidinium hydrochloride in 20 mM sodium phosphate buffer, pH 6.5. Solutions are centrifuged at 6000 x g at 4°C, the supernatant carefully removed and then re-centrifuged to obtain an optically clear solution. 

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