THE RELATION OF THE SECONDARY STRUCTURE OF PEPSIN 63 



Consequently, some of the bonds that are necessary for maintenance of 

 the enzymically active configuration are broken at lower pH values than 

 in aqueous solution. 



That the loosening of the configuration is a slight one and not similar 

 to the unfolding that is observed if other proteins are brought into contact 

 with hydrogen-bond breaking reagents is apparent from the intrinsic 

 viscosity given in Table II. Thus the intrinsic viscosity changes only from 



TABLE II 



Intrinsic Viscosity, Specific Optical Rotation, [a], and Rotatory Dispersion 

 Constant, A,., of Pepsin in Various Solvents at 25 



Composition of solvent pH* (g/ml) ' 600 m/t 400 m/i A^ 



o-i M Acetate buffer . . .4-64 3-09 63-4 178 216 



8 M Urea-acetate . . • S'Ss 3 "5? 64-1 179 218 



3 M Guanidine hydrochloride . 3'4o 3'54 62 • i 173 217 



4 M Guanidine hydrochloride . 3"4o 3'47 62-1 173 217 



* Apparent pH. 



3-09 to 3-54 (g/ml)^ if 8-0 M urea or 3-0 to 4-0 M guanidine hydro- 

 chloride is present in the reaction mixture. Moreover, as also shown in 

 Table II, the specific optical rotation, [a], and the rotatory dispersion con- 

 stant, A^., remain completely unaltered [11]. This behaviour is in contrast 

 to that found in the case of many proteins where the values of the specific 

 rotation, [a],„ of the native protein decreases by 20 to 60'' upon denatura- 

 tion [12]. Likewise, the dispersion constant. A,,, is lowered from the range 

 of 230-270 m/Li to 210 m/Li upon contact with hydrogen-bond breaking 

 reagents [13]. These changes usually reflect a major unfolding of the 

 polypeptide chain or a transition from an a-helical structure to a random 

 coil. Thus, the results obtained with pepsin further support the view that 

 no major change is brought about after short exposure to urea or guanidine 

 salts. 



If, however, as shown in Table III, the optical rotation, [7.], and the 

 rotatory dispersion constant, A,,, are measured at 55", A^, increases from 

 216 to 230 m^ii. As illustrated in Fig. 4, the increase of the rotatory dis- 

 persion constant occurs above 45", but, as indicated by the dashed lines, 

 the loss of activity accompanies or precedes the changes of A,,. It appears, 

 therefore, that certain amino acid residues, e.g., proline or serine, lock the 

 peptide chain into a configuration which confers considerable stability 

 upon the protein. At higher temperatures, however, transition to a less 

 stable configuration takes place. It is clear that in this case the chain 



