Chemical denaturing agents

Table V shows the results of this analysis for the Pn-helix fraction of several proteins denatured by heat, cold, acid, and Gdm HCl/urea. There is rather good consistency among the estimated Pn-helix contents for proteins denatured by a given agent, except for acid-denatured proteins, which show more variability. The chemically denatured proteins have 30 5% Pn-helix content near 0°C. At the other extreme, heat-denatured proteins have Pn-helix contents near 0%, with lysozyme having the highest value (8%). Although there are only two examples of cold-denatured proteins in Table V,2 they both have Pn-helix contents of about 20%. Acid-denatured proteins have Pn-helix contents ranging from 0 to 16%.

The delipidated serum lipoprotein proteins exhibit solubility differences in aqueous media. The polypeptides of HDL and the D polypeptides of VLDL are readily soluble in aqueous media, particularly in slightly alkaline low-ionic strength buffers (S28, S30). In contrast, the LDL protein does not dissolve in such buffers and, like many other water-insoluble proteins, requires denaturing agents, detergents, or suitable chemical modification. The many techniques for the solubilization of apo LDL have been reviewed recently (G15). A thorough assessment of such techniques is not possible since not all the solubilized products have been characterized. The choice of the method presently depends on the investigator s preference and experimental needs. 

The sulfur atom of methionine residues may be modified by formation of sulfonium salts or by oxidation to sulfoxides or the sulfone. The cyanosulfonium salt is not particularly useful for chemical modification studies because of the tendency for cyclization and chain cleavage (129). This fact, of course, makes it very useful in sequence work. Normally, the methionine residues of RNase can only be modified after denaturation of the protein, i.e., in acid pH, urea, detergents, etc. On treatment with iodoacetate or hydrogen peroxide, derivatives with more than one sulfonium or sulfoxide group did not form active enzymes on removal of the denaturing agent (130) [see, however, Jori et al. (131)]. There was an indication of some active monosubstituted derivatives (130, 132). 

Superdex, Superose, Sephacryl and Sepharose CL are chemically relatively inert they can be used between pH 3 and 11, and will survive more extreme conditions (pH 2-12) for short periods. They are also resistant to a range of other conditions such as 1 % (w/v) SDS, chaotropic denaturing agents (6 M guanidinium hydrochloride or 8 M urea) as well as some organic solvents (e.g formamide, DMSO, methanol, ethanol and acetone). 

Denaturation is the loss of protein function from structural change or chemical reaction. At what level of protein structure or through what chemical reaction does each of the following denaturation agents act ... 

A sufficient number of chemical groups that can be activated or modified in such a manner that they may be able to couple the affinant is a necessary condition for the preparation of specific adsorbents. The activation or modification should take place under conditions that do not change the structure of the support. No less important are the chemical and mechanical stabilities of the carrier under the conditions of attachment of the affinant, and also at various pH values, temperatures and ionic strengths, in the possible presence of denaturating agents, etc., which may be necessary for a good sorption and elution of the isolated substance. The specific adsorbent should not be attacked by microorganisms and enzymes. These stabilities are important primarily for repeated use of specific adsorbents. 

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