THE RELATION OF THE SECONDARY STRUCTURE OF PEPSIN 



6i 



readily inactivated if brought into contact with these reagents, pepsin 

 remains active after short exposure to urea and guanidine hydrochloride. 

 However, as illustrated with the aid of Fig. i, the pH of maximum hydroly- 

 sis, which in aqueous solutions is at pH i • 8, is shifted to an apparent pH 

 of 2-3 in 8-0 M urea and to pH 3-0 in 3-0 m guanidine hydrochloride. 



Further investigation of the effect of urea and guanidine hydrochloride 

 on pepsin has revealed that the enzymic activity in the presence of these 

 hydrogen-bond breaking reagents depends on a variety of factors: (i) 

 concentration of the reagent, (2) time of exposure, (3) temperature, and 

 (4) pH. 



Fig. 2. Dependence of pepsin activity on time of exposure to guanidine 

 hydrochloride at 37". 



In Fig. 2, are shown the results of measurements in which the con- 

 centration of guanidine hydrochloride was varied but the pH of the 

 reaction mixture maintained at 3-4. In 3-0 m guanidine hydrochloride, 

 pepsin is almost as active as in buffer, whereas in 6-o M guanidine hydro- 

 chloride complete inactivation occurs within 30 minutes of contact with 

 the reagent [8]. A similar behaviour of the protein is observed in experi- 

 ments in which the urea concentration is varied from 2-0 M to 8-o M [9]. 



The second factor mentioned is temperature. If, at a constant concen- 

 tration of 4-0 M guanidine hydrochloride of pH 3-4, the temperature is 

 lowered, the rate of inactivation decreases. Thus at 37 , the first-order 



rate constant, k, is 1-53 x lO" 

 k = 1-5 X 10^ X min."^ (8). 



at 



■06 



iQ-* and at 25°, 



