114 



L. G. AUGENSTINE 



can be considerably disrupted without loss in activity. They found that rever- 

 sible denaturation in 8 M urea did not cause permanent loss in activity; in 

 fact the RNase was still active in 8 M urea in which its specific viscosity was 

 8.9 as compared with 3.3 in aqueous solution. This large increase in specific 

 viscosity indicates that the so-called native configuration can be opened con- 

 siderably without destruction of activity. However, Anfinsen reports that 

 oxidation with performic acid, which disrupts the disulfide bonds, causes 

 irreversible inactivation and an increase in specific viscosity to 11.6. 



The phenomenon of complete loss in activity upon the appearance of the 

 full sulfhydryl titer has been observed in most proteins. It has also been known 

 for a number of years that different degrees of loss in characteristic activity 

 can occur. A number of workers (34, 35) have studied reversible inactivation 

 of enzymes in which it has been observed that a partial unfolding of the mole- 

 cule can occur with a rise in specific viscosity, change in the optical rotation 

 of the protein solutions, changes in solubility, etc., which upon the proper 

 treatment can be reversed. The thermodynamics for reversible denaturation 

 shown in Fig. 4 indicate that quite hkely the first step is common from protein 

 to protein since AF* is remarkably constant for all proteins. Reversible denat- 

 uration invariably shows an increase in entropy. However, AS* is not constant 

 from protein to protein but varies by a large amount as shown by the unhatched 

 areas to the right in Fig. 4. 



The author has proposed (12, 36) and discussed elsewhere in this volume 

 (37) a hypothesis involving three steps, which attempts to explain this pheno- 

 menon by ascribing the constant AF* to the initial opening of a disulfide 

 bond. This first step is followed by the rupture of a number of neighboring 

 intramolecular bonds (step 2) with a resulting opening of the molecule indicated 

 by the increase in entropy. According to the proposal, this opening of the mole- 

 cule is sufficient to disrupt the spatial arrangement of critical amino acids causing 

 loss in activity, but enough stability and configuration is retained so that under 

 the proper conditions the original native structure, or at least a structure 

 compatible with activity, can restitute. In this hypothesis the rupture of a 

 second disulfide bond (step 3) allows irreversible inactivation to proceed with 

 essentially complete destruction of the characteristic protein structure. 



A conversion (using an equivalence derived in reference (38)) has been 

 made in Fig. 4 from AS* to A/^. By assuming an average amino acid residue 



Table I 



