Protein guanidination

Number of residues/mole of protein. b Determined by protein guanidination with O-methylisourea. ... 

Folded proteins can be caused to spontaneously unfold upon being exposed to chaotropic agents, such as urea or guanidine hydrochloride (Gdn), or to elevated temperature (thermal denaturation). As solution conditions are changed by addition of denaturant, the mole fraction of denatured protein increases from a minimum of zero to a maximum of 1.0 in a characteristic unfolding isotherm (Fig. 7a). From a plot such as Figure 7a one can determine the concentration of denaturant, or the temperature in the case of thermal denaturation, required to achieve half maximal unfolding, ie, where... 

For precipitated protein, buffered solutions containing chaotropic reagents such as 0.1% SDS, 8 M urea, or 6 M guanidine or proteolytic enzymes such as pepsin may be used. However, an extended washing with buffer is required to remove SDS and guanidine. Unexpected elution behavior can occur if these reagents are not removed completely. 

The methacrylic backbone structure makes the spherical Toyopearl particles rigid, which in turn allows linear pressure flow curves up to nearly 120 psi (<10 bar), as seen in Fig. 4.45. Toyopearl HW resins are highly resistant to chemical and microbial attack and are stable over a wide pH range (pH 2-12 for operation, and from pH 1 to 13 for routine cleaning and sanitization). Toyopearl HW resins are compatible with solvents such as methanol, ethanol, acetone, isopropanol, -propanol, and chloroform. Toyopearl HW media have been used with harsh denaturants such as guanidine chloride, sodium dodecyl sulfate, and urea with no loss of efficiency or resolution (40). Studies in which Toyopearl HW media were exposed to 50% trifluoroacetic acid at 40°C for 4 weeks revealed no change in the retention of various proteins. Similarly, the repeated exposure of Toyopearl HW-55S to 0.1 N NaOH did not change retention times or efficiencies for marker compounds (41). 

Columns can be washed with solvents and solvent combinations suitable to remove adsorbed contaminants. When considering the adsorption of analytes, think not only of the diol functionality, but also of the adsorption to residual silanols. Often, the injection of small amounts (500 /d) of dimethyl sulfoxide removes contamination that has accumulated on the column. Aqueous solutions of sodium dodecyl sulfate, guanidine hydrochloride, or urea are compatible with Protein-Pak columns. 

A new protein of unknown structure has been purified. Gel filtration chromatography reveals that the native protein has a molecular weight of 240,000. Chromatography in the presence of 6 M guanidine hydrochloride yields only a peak for a protein of M, 60,000. Chromatography in the presence of 6 M guanidine hydrochloride and 10 mM /3-mercaptoethanol yields peaks for proteins of M, 34,000 and 26,000. Explain what can be determined about the structure of this protein from these data. 

Fig. 6.22 A function model of the sodium channel. P denotes protein, S the potential sensitive sensor and H the gate. The negative sign marks the carboxylate group where the guanidine group of tetro-dotoxin can be attached. (According to B. Hille)...

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