Ribonuclease scrambled

Figure 3.54. Reestablishing Correct Disulfide Pairing. Native ribonuclease can be reformed from scrambled ribonuclease in the presence of a trace of P-mercaptoethanol.

Shuffle test. An enzyme that catalyzes disulfide-sulfhydryl exchange reactions, called protein disulfide isomerase (PDl), has been isolated. PDI rapidly converts inactive scrambled ribonuclease into enzymatically active ribonuclease. In contrast, insulin is rapidly inactivated by PDI. What does this important observation imply about the relation between the amino acid sequence of insulin and its three-dimensional structure ... 

A quite different result was obtained when reduced ribonuclease was re-oxidized while it was still in 8 M urea and the preparation was then dialyzed to remove the urea. Ribonuclease reoxidized in this way had only 1 % of the enzymatic activity of the native protein. Why were the outcomes so different when reduced ribonuclease was reoxidized in the presence and absence of urea The reason is that the wrong disulfides formed pairs in urea. There are 105 different ways of pairing eight cysteine molecules to form four disulfides only one of these combinations is enzymatically active. "Fhe 104 wrong pairings have been picturesquely termed scrambled ribonuclease. Anfinsen found that scrambled ribonuclease spontaneously converted into fully active, native ribonuclease when trace amounts of 3-mercaptocthanol... 

The amino acid sequence of insulin does not determine its three-dimensional structure. By catalyzing a disulfide-sulfhydryl exchange, this enzyme speeds up the activation of scrambled ribonuclease because the native form is the most thermodynamically stable. In contrast, the structure of active insulin is not the most thermodynamically stable form. The three-dimensional structure of insulin is determined by the folding of preproinsulin, which is later processed to mature insulin. 

Haber and Anfinsen (1962) have shown that reoxidation of RNase in 8 M urea or 4 M GuHCl gives a product which contains a great number of species with incorrectly formed S—S bonds. This scrambled ribonuclease is able to regain its native structure by exposure of the material to a small amount of reducing reagent and removal of urea to allow disulfide interchange (Fig. 5.12). 

---