Denaturation, reversible

In a study of the temperature-induced reversible denaturation of the protein chymotrypsinogen,... 

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. 

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... 

Deactivation due to lyophilization by non-reversible denaturation in freeze/dry cycle... 

The DNA hybridization of an Au NP-DNA hybrid can be controlled through inductive coupling of a radiofrequency (1 GHz) electromagnetic field to the Au NP (1.4 nm), which acts as an antenna, increasing the local temperature of the bound DNA and inducing denaturation. The switch on-off of the electromagnetic field induces a reversible denaturation-rehybridization of the DNA double-helix [196]. 

The yeast enzyme is reversibly denatured in urea at concentrations of less than 2 M, probably because of dissociation into subunits.1410 At higher concentrations irreversible denaturation occurs as a result of unfolding of the polypeptide chains. 

Alkaline phosphatase can be reversibly denatured by thiol reduction in the presence of urea (88), a treatment which dissociates the dimer. Proteins purified from alkaline phosphatase-negative mutants that are antigenically related to alkaline phosphatase are readily and reversibly dissociated by acid (65). Normal alkaline phosphatase is more stable but at a lower pH, less than 3.0, it too forms monomers with release of zinc ions. However, chelating agents that remove zinc do not cause... 

The Fe—Met-80 link is also involved in the unfolding and refolding of the protein in reversible denaturation.673 The last process in the unfolding and the first process during refolding are the disruption and formation of the Fe—Met-80 linkage respectively. 

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. 

Koppelman, S.J., Nieuwenhuizen, W.F., Gaspari, M. et al. 2005. Reversible denaturation of Brazil nut 2S albumin (Ber el) and implication of structural destabilization on digestion by pepsin. JAgric Food Chem 53 123-131. 

Proteins, Reversible Denaturation of. Methods of Study and Interpretation of Data... 

Enzymatic activity, however, is not merely associated with covalent structures, but chiefly with tertiary structure which is still more difficult to determine. The crucial role of tertiary structure is proved by the fact that denaturation brings about inactivation. Even with proteins which may be reversibly denatured, such as chymotrypsin and trypsin, activity is lost as long as denaturation persists. Ribonuclease appeared for a while to be an exception, since it was still active in 8 M urea. But it was shown later that phosphate ions, at a concentration as low as 0.003 M, and polyphosphates induced in urea-denatured ribonuclease spectral changes usually associated with refolding (164). It could then be assumed that ribonucleic acid, the actual substrate, was also able to refold the denatured form and prevent inactivation in this way. In other words, even in ribonuclease, the active center is probably not built by adjacent residues in a tail or a ring, but by some residues correctly located in space by the superimposed... 

Given the limitations of the above systems, it is apparent that the optimal peptide model of a p-sheet (and a p-turn) should be as analagous to the monomeric helix models as possible. In particular, the ideal p-sheet model should be small (< 20 residues), monomeric, water-soluble, pure (composed of only p-sheets and p-turns), amphipathic (to investigate sidedness), reversibly denaturable, composed of only natural amino acids, easily synthesized and easily characterized by standard spectroscopic techniques. We believe that we have developed such a peptide model. It is based on the naturally occurring cyclic peptide gramicidin S, an antibiotic produced by the bacterium bacillus brevis (12). The schematic structure of gramicidin S as determined by X-ray and NMR studies (13, 14) is shown in Figure 1. 

The discovery that the addition of a relatively small amount of GuHCl can allow reversible denaturation will, for the first time, allow accurate determination of the thermodynamic parameters of unfolding for human aFGF. We are currently constructing a series of alanine and serine mutants at the three cysteine residues in human aFGF. DSC analyses of these mutants will allow the determination of their specific contribution to stability, separate from their effects upon irreversible denaturation. 

---