Renaturation/reoxidation

Figure 11. Schematic illustration of the fate of protein S-S bonds during reduction and unfolding followed by renaturation and reoxidation (16)...

Chromophore modifications 1) Reduction with borohydride (modified from ref. 10). A solution of PC or isolated subunits (chromophore concentration 7-21/iM, 0.9M potassium phosphate, pH 7, 8M urea) was treated with NaBH (170 mM). After complete reduction (spectrum, 45 min) excess reductant was destroyed by glucose. For reoxidation experiments, the samples (lOOmM potassitim phosphate, pH 7, containing 70% ammonium sulfate to prevent protein degradation) were allowed to stand at room temperature in the dark for up to nine days. This was followed by recombination (if not yet done), denaturation (8M urea in lOOmM potassium phosphate, pH7) and renaturation as described below. 2) Photobleaching of the o-subunit (8/xM protein, 100 mM phosphate, 8M urea, pH 7.5) was... 

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-... 

A crucial experiment will be to reoxidize reduced RNase in glycerol, and see if the native disulfides are regenerated. It will also be valuable to see if non-disulfide proteins are renatured in these solvents. 

The presence of trace of jS-mercaptoethanol accelerated the renaturation process of RNase and lysozyme (Haber and Anfinsen, 1962 Epstein et al, 1962 Epstein and Goldberger, 1963). In the case of RNase, j8-mercaptoethanol catalyzed the disulfide interchange in incorrectly reoxidized enzyme. The effect of )8-mercaptoethanol on the reactivation of lysozyme is shown in Fig. 5.13. It prevents aggregation of the protein and is required to obtain the maximal rate of reactivation of lysozyme, in contrast with RNase where dilution alone is able to overcome the rate limiting effect of aggregation. 

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