Guanidine chloride

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). 

Effect of guanidine chloride concentration on / -galactosidase activity... 

In the following experiment, we will study /3-galactosidase denaturation by guanidine chloride following OD of PNP at 405 nm. Enzyme denaturation induces a loss in its activity, which will be observed by the decrease in the OD. 

Thus, it is possible to calculate the equilibrium constant at each guanidine chloride concentration using Equation (4.19). Keq = f ([guanidine]) yields the graph shown in Figure 4.12. 

In our experiments, although equilibrium between native and denatured forms of /i-galactosidase exists at all guanidine chloride concentrations, at high concentrations the enzyme will be in one form, denatured, and thus there will be no kinetic equilibrium. 

The AG0/ variation with guanidine chloride concentration can be described by the following equation ... 

Figure 4.14 Variation of AC0 of the enzymatic reaction with /J-galactosidase as a function of guanidine chloride concentration.

Figure 4.16 Variation of AG0 with guanidine chloride concentration at25°C.

Effect of guanidine chloride on hydrolysis kinetics of p-nitrophenyl-fi-D-galactoside... 

Figure 4.17 Activity of jS-galactosidase with time in the presence of 7 mM guanidine chloride. Analysis of the exponential decrease as the sum of two exponentials yields decay times of 2.9 and 14.5 min.

Figure 4.17 displays -galactosidase activity in the presence of 7 mM of guanidine chloride as a function of time. Analysis of the decay curve yields two decay times equal to 2.9 and 14.5 min. These times reveal mainly that the denaturation process of a protein does not occur in one simple step. The structure and dynamics of the protein should play an important role in the accessibility of the guanidine chloride to the amino acids and so to the unfolding process. 

Protein charges that maintain the folded structure will be neutralized the electrostatic mteraction with guanidine chloride inducing by that an unfolded protein Also, mteraction occurs with the amino acids of the inner core of the protein inducing by that an irreversible unfolded state in presence of the denaturant. Equation 1.13 gives the relation that exists between the intrinsic viscosity of proteins (p) in presence nf 6 M guanidine chloride and the number (n) of amino acid residues per protein chain (Tanford elal. 1967). 

In the following example, we have studied denaturation of P galactosidase by guanidine chloride following the optical density of PNP at 410 nm. Denaturation of the enzyme will induce a loss in the enzyme activity, which will be observed by the decrease of the optical density at 410 nm. 

Figure 1.21. Effect of Guanidine chloride concentration on the activity of p-galactosidase.

With the denaturing method, the sample is dissolved in buffer containing urea or guanidine chloride. Typical compositions of such a lysis buffer would be 8 M urea, 1 M NaCl, 10% glycerin pH 8.0 or 6 M guanidine chloride, 100 mM NaH2P04, and 10 mM Tris-Cl pH 8.0. In the case of membrane proteins, you could still add 0.5% NP-40 or TRITON-X-100. 

Fig. 8 (a) Comparison of the force curve of ssDNA obtained in DEB and the QM-FRC fitting curve, (b) Comparison between the smoothed force curve of ssDNA obtained in aqueous solution and in DEB. (c) Comparison between the force curves of ssDNA obtained in various aqueous media and QM-FRC fitting curve, (d) Comparison between the force curves obtained in aqueous guanidine chloride and DEB. Figure reproduced with permission from [31]... 

M guanidine chloride, 10 mM dithioihreitol, 50 mM Tris-HCl 150mM NaCl, 50mM Tris-HCl (pH 7.4), 35 °C... 

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