Of guanidine hydrochloride

Fig. 41. LS plots for solutions of bovine serum albumin (concentration c2) in aq. 6 M guanidine hydrochloride, obtained at constant molarity C3, constant chemical potential n3 and constant molality m3 of guanidine hydrochloride. The resultant molecular weights yielded for this sample are given above each plot15)...

Blaber, S.I., J.F. Culajay, A. Khurana, and M. Blaber. 1999. Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride. Biophys J 77 47CM-77. 

Estimated from thermal melting in the presence of different concentrations of guanidine hydrochloride. 

Note that in the case of the reaction of guanidine hydrochloride with perfluoro-5-azanon-4-ene one obtains a mixture of products 102 and 103. Compound 103 is the product of further reaction of perfluoro-5-azanon-4-ene with 102. It may be assumed that 103 is formed according to the following scheme ... 

The thiols in native lipoamide dehydrogenase are remarkably unreac-tive with other reagents only one thiol is at all reactive with DTNB or iodoacetate (61). Formation of the TNB-enzyme mixed disulfide is greatly increased by low concentrations (0.7 M) of guanidine hydrochloride (166). Its modification is associated with the destabilization of the enzyme in 1 Af guanidine hydrochloride which results in the slow reaction of 6 additional thiols. If the denaturant and excess DTNB are removed when the single thiol has reacted, the spectrum of enzyme-bound FAD is unmodified and the enzyme retains almost full activity. It is concluded that the thiol and the FAD are remote from one another in the protein (166). 

Fig. 28. The shift of resonance signals of Cyclo-(Gly-Glu(OBzl)-Gly)2 in DMSO-d with the addition of guanidine hydrochloride. R represents moles of guanidine hydrochloride per unit mole of peptide bond of Cyclo-(GIy-Glu(OBzl)-Gly)2...

Moles of guanidine hydrochloride per unit mcrfe oi pepUe bond Cycoupling constant from NH qgnals. 

D, ), Glu(OBzl)-NH. Full symbols represent the peptide NH signal in the presence of guanidine hydrochloride R 2)... 

There is a simple way to avoid these problems. One can define a pH scale in a completely arbitrary manner relative to the emf of a suitable cell. One can then relate the pH on this scale to an arbitrarily defined activity of hydrogen ions, simply be setting pH = —log an+. The dissociation constants of model compounds can then be determined in terms of this arbitrary scale. This method has been used by Donovan et ai. (1959) for protein titrations in concentrated aqueous solutions of guanidine hydrochloride and of urea, and by Sage and Singer (1962) for titrations in ethylene glycol. 

Regardless of whether the protein is subjected to pH values <4.0 or >9.0, is heated above 50°C, exposed to low concentrations of guanidine hydrochloride, or subjected to Ca(II) removal from the N form, all usually appear to involve dissociation of Ca(II) in some way. 

Diamino-3-chloro-5-hydrazinocarbonylpyrazine with nitrous acid at 50-55° formed 2,6-diamino-3-azidocarbonyl-5-chloropyrazine, which, refluxed with a solution of guanidine hydrochloride and sodium isopropoxide in propan-2-ol, gave 2,6-diamino-3-chloro-5-guanidinocarbonylpyrazine and many similar preparations were reported (1371). 2-Amino-5-chloro-3-hydrazinocarbonylpyrazine treated with nitrous acid gave 2-amino-3-azidocarbonyl-5-chloropyrazine, which was pyrolyzed to 5-chloro-2-hydroxyimidazo(4,5-Z)]pyrazine (1226). 

Figure 6. Equilibrium denaturation isotherms for Pal-HI (solid symbols) anrt HT t symbols) in pH 7.5 buffer containing 20% EtOH determined from the ellipticity at 224 nm°The fraction of protein unfolded is plotted as a function of guanidine hydrochloride concentration...

For QD and fluorescence spectroscopic analyses of (o-[l-Sei46]Aga-TK and co-[D-Ser46]Aga-TK, peptide samples were prepared by freshly dissolving the lyophilized peptides at a concentration of 150 ng/ml in Dulbecco s phosphate-buffered saline, pH 7.4, in the presence or absence of guanidine hydrochloride. CD spectra were recorded with a Jasco J-720WI spectropolarimeter at room temperature using a 0.1 cm path-length cell. In aU cases, the buffer base-line spectrum was subtracted, and the results were expressed in terms of the mean residue ellipticity... 

The apparent molecular mass of D-Ser toxin was dramatically increased by the addition of guanidine hydrochloride to the elution buffer, although that of the L-Ser toxin was not altered by the denaturing reagent. In the presence of 5.2 M guanidine hydrochloride, the D-form toxin was eluted at the same position as the L-form toxin and the apparent molecular masses of the two toxins were estimated as 6 kDa based on calibration with the standard proteins. CD and fluorescence spectroscopic analyses revealed that the two toxins were unfolded and lost their secondary and tertiary structure in 5.2 M guanidine hydrochloride at pH 7.4, as described below. It, therefore, appears that the D-Ser toxin forms a compact folded structure, whereas the L-Ser toxin has a relatively unfolded or extended structure. 

Figure 3. CD spectra of co-[D-Ser ]Aga-TK and to-[L-Ser ]Aga-TK in phosphate-buffered saline, pH 7.4, in the presence or absence of guanidine hydrochloride.

Fig. 4. Guanidine denaturation of apolipoprotein E as assessed by circular dichroism. The molar ellipticity (0) of apoES (0.1 mg/ml in 20 mAf phosphate, pH 7.4, 1 mAf dithiothreitol) as a function of guanidine hydrochloride concentration was determined at 25°C. From Wetterau et al. (1988).

Fig. 2. Reverse-phase HPLC profile of r-hGMF-beta. (A) Pure r-hGMF-beta without treatment. (B) After treatment with 10 mM DTT in presence of 6M guanidine hydrochloride. Note identical retention time in each case. (C D) Time course of oxidative refolding of r-hGMF-beta and separation of isoforms by reverse-phase HPLC. Pure r-hGMF-beta was dissolved in O.IM sodium phosphate and incubated at room temperature with reduced and oxidized glutathione in the presence of guanidine hydrochloride, as described in the text, for 4 h (C) and 8 h (D). (Adapted from ref. 17)...

It was originally suggested that the links between the basic units are through intermolecular disulfide bridging between cysteine residues in the naked end r ons. Reduction of mucins by thiols produced the M, 500,000 forms (see, for example. Refs. 26 and 36). However, Creeth observed a variety of forms in the 0.5-2.0 X 10 r on, and subsequent observations on cervical and other mucins by Carlstedt and Sheehan S yielded, in the presence of guanidine hydrochloride (Gu-HQ), species having forms M, 2 X 10 which they referred to as... 

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