62 



GERTRUDE E. PERLMANN 



The next point to be discussed is the effect of pH on the rate of in- 

 activation. In Fig. 3, is given a comparison of the effect of 8-o m urea and 

 4-0 M guanidine hydrochloride if pepsin is exposed to the reagent for i 

 hour at 37°. Three points become apparent: (i) The range of maximum 

 stabihty of pepsin, which in aqueous solution extends to pH 5-5, is 

 narrowed to pH 3-0 to 3-5 in 4-0 m guanidine hydrochloride and to pH 

 3-3 to 4-3 in 8-0 M urea, respectively. (2) Although denaturation of 

 pepsin at low pH values, i.e. i -5, has been reported by Northrop [10], the 

 loss of activity in the presence of hydrogen-bond breaking reagents 

 proceeds rapidly below pH 3 -o, and the formation of low molecular weight 



100 



80 



-60 



-40 



-20 



O 0) 



o^ 



-c E 

 - ^ 



% O 



12 3 4 5 6 



pH 



Fig. 3. Effect of various solvents on pepsin. Time of incubation: i hour at 



37 • 



peptides, resulting from the action of intact pepsin on the denatured 

 material, closely parallels the rate of inactivation. (3) At pH values more 

 alkaline than 6-o, pepsin loses its activity spontaneously without the forma- 

 tion of non-protein material. In the presence of guanidinium ions or urea, 

 this rapid loss occurs at more acid pH values. Moreover, it is of interest 

 to note that although the pH zone in which inactivation takes place depends 

 on the reagent and its concentration, the maximum rate of inactivation, 

 within a small pH interval is independent of the nature of the solvent. One 

 may, therefore, conclude that intramolecular bonds of the same nature 

 are sensitive to the hydroxyl ions of the medium. Thus, in the pH range 

 of 3-5 to 5-5 the main effect of the hydrogen-bond breaking reagents 

 consists in loosening the secondary structure of the pepsin molecule. 



