Modification of pepsin by autodigestion 



GERTRUDE E. PERLMANN AND 

 MARY J. MYCEK* 



The Rockefeller Institute for Medical Research, New York, U.S.A. 



In the work on protein structure one objective has been the discovery and 

 use of enzymes that attack linkages in specific configuration thus leaving 

 the protein substrate relatively intact. Although the action of such enzymes 

 may consist in as little as the cleavage of one bond — or the removal of a 

 certain group of the molecule — considerable changes in some of the pro- 

 perties of the protein may occur. ^ Thus it has been possible to demonstrate 

 that the enzymic removal of the pepsin-phosphorus does not impair the 

 proteolytic activity of the protein although its electrophoretic mobility is 

 altered. In contrast to these results it is of interest that through the action 

 of 'pepsin on pepsin' a modified form of the protein can be obtained. Here, 

 the physico-chemical properties such as the electrophoretic behavior and 

 the molecular weight have not changed appreciably but the relative specific 

 enzymic activity is higher than that of the parent substance. 



As shown by Steinhardt,^ pepsin, if stored in 1-0 to 60 m urea at 3°C 

 for one to fifteen days, retains its enzymic activity. However, a marked loss 

 of proteolysis occurs, if pepsin is exposed to 8-0 M urea at temperatures 

 above 20°C.^ Thus, the activity of a 0-5 per cent pepsin solution kept in 

 urea-acetate of pH 5-3 and 0-07 ionic strength for 24 hours at 25°C, is 

 reduced to 40 per cent of its initial value, whereas at 37°C almost complete 

 inactivation takes place. The rate of inactivaiion is a function of the urea 

 concentration, the pH and the ionic composition of the solvent. In addition 

 to the loss of enzymic activity the absorption maximum of pepsin in the 

 ultraviolet is shifted from 2780 Â to the shorter wave length of 2760 Â after 

 treatment with urea. It emerges from these results that the action of the 

 urea on pepsin consists in the rupture of hydrogen-bonded phenolic hydroxyl 

 groups of the tyrosine residues. The protein unfolds slightly and a number 

 of peptide bonds hitherto not accessible to enzymic hydrolysis are now 

 exposed and rapidly hydrolyzed. The view that an enzyme-catalyzed auto- 

 lysis is the underlying phenomenon is further supported by the fact that 

 exposure to urea produces an increase in the solubility of pepsin in hot 

 10 per cent trichloroacetic acid. The formation of non-protein material is 

 most marked in the pH range of 4-0 to 5-5. 



* Public Health Service Postdoctoral Fellow of the National Cancer Institute, National 

 Institutes of Health. 



