11] STRUCTURE AND ACTIVITY OF PAPAIN 197 



More recently, a method has been developed for separating chromato- 

 graphically, inactivated aminopeptidase from mercuripapain or from de- 

 graded mercuripapain.^^ The chromatography is shown in Fig. 5. The main 



60 80 



TUBE NUMBER 



Fig. 5. Chromatographic separation of the proteins in an inactivated preparation of 



leucine aminopeptidase from mercuripapain (upper figure) or degraded mercuripapain 



(minus approximately 50 residues) (lower figure). A 1-3 x 40 cm. column of IRC-50 was 



used at pH 6-3, initially with 0-1 m phosphate buffer and then with 0-2 m phosphate. (Hill 



and Smith, unpublished.) 



feature of the chromatography on IRC-50 is that inactivated aminopepti- 

 dase and contaminating proteins of the aminopeptidase preparation are 

 readily eluted with 0-1 m phosphate buffer at pH 6-3, whereas the mercuri- 

 papain or degraded material is quantitatively absorbed. Phosphate buffer 

 at 0-2 M is then used to displace the mercuripapain. Although neither the 

 inert mercuripapain nor the degraded material shows symmetrical peaks, 

 it is evident that there is excellent agreement between the curves for relative 

 activity and for ninhydrin color. This, in effect, demonstrates the absence 

 of significant amount of inert material. 



The contents of the tubes containing the degraded material were pooled 

 and worked up by conventional methods. The mixture was then hydrolyzed 

 with 6N-HC1 and oxidized with performic acid. Unfortunately, this pro- 

 cedure destroys the tryptophan and much of the tyrosine, but values were 

 obtained for the other amino acids. Values for tyrosine and tryptophan will 

 have to be estimated in other experiments. 



In order to compare the results of Experiment II on the hydrolysate of 

 residual papain with the analyses for the liberated amino acids, it has been 



