Denaturation solute concentration

Temperatures above (and sometimes below) the normal range will cause thermally unstable proteins to unfold or denature . High concentrations of solutes, extremes of pH, mechanical forces and the presence of chemical denaturants can do the same. A fuUy denatured protein lacks both tertiary and secondary structure, and exists as a random coil . In most cases denaturation of proteins is irreversible. 

Renaturation. The effects of i) reduction of the disulfide bond of p-lactamase, ii) renaturation buffer pH, iii) concentration of protein, iv) GuHCl in the denaturing solution and finally v) sucrose concentration on the reversibility of the unfolding transition were investigated. 

The protein is diluted with denaturation solution if necessary to reach a protein concentration of 10 mg/mL. 

DNA denaturation. Solutions (100 pi) were prepared with final concentrations of DNA (50 pg/ml), formamide (4.1%), and formaldehyde (0.1%) in 0. lxTE buffer, incubated at 52 °C for 15 min, and cooled rapidly on ice. Stored at -20 °C (freezing the samples at least once was essential). The calf thymus DNA calibration samples were handled in the same way. 

Effect of Mineral Salts. As cellular water is frozen, mineral salts and soluble-organic substances become concentrated in the remaining unfrozen phase. This increase in solute concentration, with corresponding changes in ionic strength and pH, is believed to affect dissociation and/or denaturation of proteins (1,2,62,96-98). Experiments by Fukumi et al. (99) support this theory. They found that freeze denaturation of washed actomyosin from Alaska pollack muscle was accelerated by the presence of Ca2+, Mg2+, K+, and Na+ ions and reduced by their removal. 

A Stock solution of the protein dialysed against (or, if lyophilized, dissolved in) the same buffer. The concentration will depend upon the optical techniques being used. For CD studies, a 50-200 j,m protein stock solution should allow a minimal volume to be added to the denaturant solution to give a working concentration in the 5-20 (jlm range Pipettes... 

A fresh, filtered solution of 20 mM NaHjPO.), pH 7.0 containing the desired final concentration of the protein (e.g. prepared by addition of an aliquot from a concentrated protein stock solution). Prepare approximately 2.0 ml of this zero-denaturant solution... 

A solution of 8 M urea in 20 mM NaH2P04. pH 7.0 also containing the same final protein concentration (firmn addition of an aliquot of concentrated stock). This is the concentrated denaturant solution. Depending on the midpoint urea concentration for 50% unfolding, one will need from 2 ml (for a low midpoint) to 6 ml of this solution (for a hi midpoint) Q.uartz cuvettes Pipettes... 

Remove an aliquot (e.g. 50-200 til) of the solution fiom the cuvette and then add the same volume of the concentrated denaturant solution to the cuvette. 

The cytochrome c case is noteworthy becanse at NEEs, weU-resolved CVs are obtained in diluted solutions of the protein both with and without promoters such as 4,4 -bipyridyl typically used for promoting cytochrome c electrochemistry (99-101). These promoters are generally required to avoid adsorption/denaturation (83, 102, 103) of cytochrome c on the Au surface. However, such an adsorption is concentration dependent so that lowering the cytochrome c solution concentration below the adsorption limit (possible at NEEs thanks to their lower detection limit) can overcome adsorption-related problems. A similar situation has been reported for the adsorption of some organic dyes such as the phenothiazines (69). 

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... 

Product recoveiy from reversed micellar solutions can often be attained by simple back extrac tion, by contacting with an aqueous solution having salt concentration and pH that disfavors protein solu-bihzation, but this is not always a reliable method. Addition of cosolvents such as ethyl acetate or alcohols can lead to a disruption of the micelles and expulsion of the protein species, but this may also lead to protein denaturation. These additives must be removed by distillation, for example, to enable reconstitution of the micellar phase. Temperature increases can similarly lead to product release as a concentrated aqueous solution. Removal of the water from the reversed micelles by molecular sieves or sihca gel has also been found to cause a precipitation of the protein from the organic phase. 

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