180 GERTRUDE E. PERLMANN AND MARY J. MYCEK [10 



Since at any given stage of the urea inactivation not more than 70 per 

 cent of the trichloroacetic acid soluble material is dialysable, samples of 

 pepsin treated with 8-0 m urea at 37°C for 6, 12, 18 and 24 hours, respectively, 

 were dialyzed and analyzed electrophoretically. As indicated in Fig, 1, the 



Time of Relative specific Zlectpophopetic 



exposure to urea proteolytic activity composition 



in hours pep unit weight 



100 100 % 



88% P 



6 51 1% p. 



h.h ->-. 5% pj 



75 %P 

 12 26 14% p, 



11 % p, 



67 %P 

 18 15 17% p, 



16% Pj 



63 %P 

 2-* e 20 % p, 



17% pj 



Fig. 1. Tracings of electrophoretic patterns of 7% pepsin solutions in sodium acetate 

 buffer of pH 4-6 and 0-1 ionic strength. Electrophoresis was carried out for 5400 sec. at 

 a potential gradient of 6-4 volts per cm. 



loss of activity is accompanied by the appearance of two new components, 

 Pi and p2, formed at the expense of the original protein, P. The relative 

 specific activity of the mixture which has been exposed to urea for 24 hours 

 is only 8 per cent of the original. On removal of the low-molecular weight 

 material by dialysis, the relative enzymic activity of the non-dialyzable frac- 

 tion, however, is similar to that of the original protein. Hence, the question 

 arises whether the residual activity is due to the presence, in the reaction 

 mixture, of some unchanged pepsin. 



To test this hypothesis the electrophoretic components of pepsin after 

 treatment with urea at 37°C for 24 hours, were separated by zone electro- 

 phoresis on a Geon resin. As shown by Fig. 2, the distribution of the 

 electrophoretic components is similar to the pattern obtained in free elec- 

 trophoresis (Fig. 1). The relative concentration of Pi and P2, however, had 

 decreased considerably owing to the fact that this sample had been pre- 

 dialyzed against a hydrochloric acid-glycine buffer of pH 2-5 and 0-1 ionic 

 strength prior to the dialysis against the acetate buffer used in the electro- 

 phoresis experiment. As indicated in Fig. 2, only the major component of 

 the mixture with a mobility similar to that of pepsin had proteolytic activity. 



Although the sedimentation constant of the active component, ^'20 = 

 2•8lXlO-^^ is only slightly lower than that of pepsin, S2o = 2-9qX\0-^^,* 

 the following differences have been established: The relative specific activity 

 per unit nitrogen of the modified protein is 40 to 50 per cent higher than 



* The authors are indebted to Dr R. Trautman for the ultracentrifuge measurements. 



