STRUCTURAL BASIS OF RIBONUCLEASE ACTIVITY 



139 



CHROMATOGRAPHV ()(E-64) OF IIMITED PEPSIN DIGEST OF BIBONUCLEASE 



r- 



0< 

 



-r 



T 



-r 



T- 



T- 



T — r 



T- 



»«»»»»» mw 



S 



10 20 30 40 iO 60 70 80 90 



TUBE NUMBER 



IJO 130 l«0 150 160 I'O 160 190 JOO JIO 220 330 Z'O 250 



IBE NUMBER 



Fig. 10. Chromatography (XE-64) of limited pepsin digest of ribonuclease 



has been observed by Weil and Seidles (1955) that the photodestruction of a 

 single histidine residue leads to inactivation. Since two of the four histidine resi- 

 dues of ribonuclease are located within the last tifth of the molecule and in view 

 of the present studies on pepsin digestion and urea treatment of ribonuclease, 

 we must tentatively conclude that part of the active center of ribonuclease is 

 located near the C-terminal end of the molecule and may involve a relatively 

 simple constellation of amino acids. It is important, however, to emphasize that 

 activity cannot be due to a simple amino acid sequence alone in view of the 

 inertness of the oxidized enzyme. Our present picture of the arrangement of 

 S — S bridges suggests that the active center may involve peptide sequences 

 located in two or more positions along the protein chain which are separated in 



2.2 



2.0 



.W 1.8 



1.6 



1.4 



1.2 



- NATIVE RNASE 



T 1 r 



OXIDIZED 

 DERIVATIVE 



OXIDIZED 

 RNASE 



I L 



_L 



0.50 1.00 1.50 



PERCENT CONCENTRATION 



Fig. 11. Sedimentation studies on the inactive derivative of ribonuclease produced 

 by pepsin digestion before and after oxidation of the disulphide bridges. The experi- 

 mental points on the upper curve were obtained on the derivative before performic 

 acid oxidation. The experimental points from the center curve are from studies on this 

 derivative after oxidation. 



