Guanidine hydrochloride denaturing agent

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... 

The intrinsic viscosity of native ribonuclease is very low. Harrington and Schellman (247) reported 3.3 ml/g at neutral pH in 0.1 M KC1. Buzzell and Tanford (265) found values of 3.3-3.5 ml/g over the entire pH range from 1 to 11 and ionic strengths from 0.05 to 0.25 M. This value increases dramatically on denaturation even without oxidation or reduction of the disulfide bonds to 8.5 ml/g (266). In the presence of reducing agents and 6 M guanidine hydrochloride the value is 16.0 ml/g (267). 

Although guanidine hydrochloride is a potent chaotropic agent, its use for RNA isolation has not been as popular as that of guanidine thiocyanate. The reasons may be that it needs to be used at a much higher concentration to be an effective protein denaturant. The method described here is a modification of methods described in Refs. 15 and 16. 

With an efficient denaturing agent such as guanidine hydrochloride (Tanford etal. 1966, 1967) and in the presence of 2-mercaptoethanol, a more uniform environment could be reached and hence a normalization of the fluorescence parameters, a condition which has to be met before applying the process for quantitative analytical purposes. 

Chaotropic agents guanidine hydrochloride use for study of protein denaturation GTIC is considered to be more effective than GuCl GTIC used for nucleic acid extraction. 

It is apparent that while incubation in solutions of low guanidine hydrochloride concentration may sometimes cause inactivation (carbonic anhydrase), it may also promote pro r refolding (chymotrypsinogen). In addition, replacement of a strong denaturant with a weaker one is an approach which can be used to successfully refold polypeptide chains. These data suggest that the rate of removal of the denaturant agent can influence yield of active protein. In this respect, time is an important refolding parameter. 

Guanidine hydrochloride at a concentration of at least 2 M dissociates the bovine enzyme, although a protease, which may contaminate some commercial preparations of the enzyme, is active under these conditions (104). A cooperative transition occurs as the concentration of urea is increased through 4 M, resulting in dissociation into subunits (68). The NADP-GDH s from Salmonella (36) and E. coli (73) are more resistant to these denaturing agents activity is retained in the presence of 4 M urea or 2.5 M guanidine hydrochloride. 

Additional steps are necessary if the initial protein sample is actually several polypeptide chains. SDS-gel electrophoresis under reducing conditions should display the number of chains. Alternatively, the number of distinct N-terminal amino acids could be determined. For a protein made up of two or more polypeptide chains held together by noncovalent bonds, denaturing agents, such as urea or guanidine hydrochloride, are used to dissociate... 

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