Reversible renaturation

Ward, W. W., and Bokman, S. H. (1982). Reversible denaturation of Aequorea green-fluorescent protein physical separation and characterization of the renatured protein. Biochemistry 21 4535-4540. 

An alternative to the injection method for importing enzymes into a microemulsion is the phase transfer method. In this method, a layer of an aqueous enzyme solution is located under a mixture of surfactant and oil. Upon gentle shaking, the enzyme is transferred into the reverse micelles of the hydrocarbon phase. Finally, the excess of water is removed and the hydrophobic substrates can be added. The main advantage of this method is that it ensures thermodynamically stable micro emulsions with maximum water concentrations. However, the method is very time consuming. The method is often applied in order to purify, concentrate or renaturate enzymes in the reverse micellar extraction process [54-58]. 

In subsequent years, much evidence has been adduced to support this mechanism. Alkaline phosphatase and, by analogy, other serine enzymes, are directly phosphorylated on serine serine phosphate is not an artifact (Kennedy and Koshland, 1957). In the presence of nitrophenyl acetate, chymotrypsin is acetylated on serine, and the resulting acetylchymotrypsin has been isolated (Balls and Aldrich, 1955 Balls and Wood, 1956). Similarly, the action of p-nitrophenyl pivalate gave rise to pivaloyl chymotrypsin, which could be crystallized (Balls et al., 1957). Neurath and workers showed that acetylchymotrypsin is hydrolyzed at pH 5.5, but that it is reversibly denatured by 8 M urea the denatured derivative is inert to hydrolysis and even to hydroxylamine, whereas the renatured protein, obtained by... 

The tertiary structure of a globular protein is determined by its amino acid sequence. The most important proof of this came from experiments showing that de-naturation of some proteins is reversible. Certain globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity if returned to conditions in which the native conformation is stable. This process is called renaturation. 

A good example of its use applied to a protein associated with food chemistry is that of P-lactoglobulin. Ikeuchi et al. (2001) used ANS as a probe to follow P-lactoglobulin de-naturation under high pressure and its subsequent renaturation on release of pressure (Fig. B3.6.11). The denaturation was shown to be completely reversible at pH 2 but not at neutral pH, explaining why whey protein isolates subjected to high pressures form a gel at pH 7 but not at acid pH. 

An alkali-detergent solution is used during the recovery of plasmid DNA. The alkaline pH causes the chromosomal DNA to be irreversibly denatured while the plasmid is reversibly denatured. The mixture is subsequently neutralized by the addition of a suitable reagent. At neutral pH, the plasmid DNA renatures and remains in solution while the denatured chromosomal DNA precipitates, forming a complex network with other materials, such as proteins and cell debris. The precipitated material flocculates and transforms into a porous gel over a period of 1 to 2 hours. The gel slowly floats to the surface, leaving behind in the solution the plasmid DNA and the fine particulates. 

Chemists have long appreciated that a protein s primary amino acid sequence determines its three-dimensional structure. It has also been known for some time that proteins are able to carry out their diversified functions only when they have folded up into compact three-dimensional structures. The protein-folding problem first gained prominence in the 1950s and 1960s, when Christian Anfinsen demonstrated that ribonuclease could be denatured (unfolded) and renatured reversibly. 

After renaturation, the majority of the recombinant chemokines are easily quantified by UV spectroscopy, although there are examples of chemokines such as NAP-2, which do not possess an aromatic amino acid that serve as chromophores. In this case, quantification is achieved by comparison of the peak height on reverse phase HPLC analysis to that of a known concentration of another chemokine. They can then be lyophilized after a change of buffer into a trifluoroacetic acid or acetic acid solution, which facilitates their storage as lyophilized powders. It is important that they are redissolved in water, before dilution into buffer or medium. Their handling is easy and rapid, as they are instantly soluble at concentrations as high as 1 mM if necessary, in aqueous solutions. 

Denaturation was early observed to be a reversible process. Indeed, Anson (1 ) observed 35 years ago that hemoglobin could be heat denatured in a variety of ways and could be converted back to a state which had all the characteristics of its original native state, as determined by methods available at that time. Almost all studies of protein denaturation now revolve around not only the denaturation itself, but also its renaturation perhaps renaturation is a more interesting and provocative field for modern research. 

The reversibility of the reductive scission of disulfide linkages has also been discussed in a previous section. Such a reversal is intimately related to the renaturation process because the thiol groups must accept their correct original partners in the formation of the disulfide linkage. In mixtures of different proteins or with other substances, the correct pairing may be prevented. 

Refolding yields can be improved by utilizing defensive reversible denaturation". This technique has been shown to be effective in restoring the activity of denatured interferon preparations (103). It was observed that interferons denatur by a variety of techniques can be partially refolded by incubation in low concentrations of urea. When SDS was added to the urea-interferon solutions prior to renaturation, full activity of the interferons could be restored. Stewart et al. (103) interpreted the stabilizing effect of SDS as "defensively" denaturing the protein solution. 

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. 

Determination of the Urea Dependence of Renaturation Yields. Renaturation solutions were prepared by mixing inclusion bodies, 7.5 M urea stock solution, and water to a final mBST concentration of 9.5 mg/mL at the desired urea concentration and a total volume of 100 mL. The solution pH was adjusted to 11 with 2.5 M NaOH and magnetically stirred in an open container to promote the air oxidation of the reduced mBST. When the oxidation was complete, usually after 2 days as judged by reverse-phase HPLC, the samples were analyzed for monomeric mBST. Samples oxidized at urea concentrations below 4 M were either dissolved at higher pH and adjusted back to pH 11 or dissolved at higher urea concentration and diluted back to the desired concentration. 

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