Ribonucleases denaturation, renaturation

Ribonuclease denatured by 8 M urea and a mercaptan (RSH a reagent that reduces disulfides to sulfhydryl groups) can be renaturated by removing the urea and RSH and air oxidizing the reduced disulfides. 

Urea is used to denature ribonuclease, and mercaptoethanol (HOCH2CH2SH) to reduce and thus cleave the disulfide bonds to yield eight Cys residues. Renaturation involves reestablishment of the correct disulfide cross-links. 

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. 

At first glance, the notion that the assistance of a molecular chaperone may be required for the folding and/or assembly of other proteins appears to be at variance with the work of Anfinsen (5), who demonstrated that bovine pancreatic ribonuclease can be denatured and consequently renatured in vitro, in the absence of other cofactors. This experiment has been repeated since with many other proteins, including Rubisco (4) thus, it has been assumed that the primary sequence is able and sufficient to direct the correct self-folding of all proteins into their functional tertiary structure. 

The one-gene-one-enzyme concept did imply that the primary structure of the peptide determines the secondary, tertiary, and quaternary structure, and this was established by Anhnsen (1973) by an analysis of the mutant ribonuclease and by the study of chemical modification as well as the denaturation and renaturation kinetics of this enzyme (Anhnsen 1973). 

The secondary and tertiary structure of a peptide is a function of the primary structure or the amino acid sequence of the peptide. This fact was established by Christian Anfinsen based on the denaturation or unfolding of an enzyme ribonuclease in the presence of urea and the renaturation or folding of the same enzyme after removal of the denaturing substance, i.e., urea. It is important to understand the secondary structure of a protein as a prelude to understanding of the tertiary structure and the function of proteins. It is important to know the rules that proteins follow to assume a 3D structure because of their roles in cellular function and their manipulation in biotechnology and drug design. 

Fig. 1 is a diagrammatic representation of a) the reductive denaturation of ribonuclease in 8 M urea with thioethanol (2-mercaptoethanol), b) its reoxidation in 8 M urea to one of the 105 possible disulfide bridge isomers, and c) its renaturation to the enzymically active form in urea-free medium, by disulfide exchange in the presence of traces of thioethanol. Formation of completely disordered structures results in irreversible denaturation, e. g. heat denaturation of ovalbu-... 

Protein folding the folding of a random coil polypeptide into its native structure, i.e. its 3-dimensional, biologically functional structure, also known as the native conformation. Loss of this native structure is known as denaturation, and the re-establishment of native structure is known as renaturation. (For the reductive denaturation and oxidative renaturation of pancreatic ribonuclease, see Protein). 

Among all possible configurations, the active enzyme conformation is assumed to be thermodynamically the most stable in situ. Experimental evidence to validate this assumption came from reversible denaturation studies on enzymes. For example. ribonuclease may be completely unfolded in urea solution after breakage of four native intramolecular disulphide bridges. Afterwards, when ribonuclease is brought to renature in the proper conditions, the native disulphide bonds are restored, and the protein recovers its catalytic activity. This case must be regarded, however, as a very favourable one because in most instances renaturation is... 

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