198 EMIL L. SMITH, ROBERT L. HILL AND J. R. KIMMEL [11 

 assumed, as found earlier (Table 7), that only one residue of cysteic acid is 

 found among the oxidized free amino acids and five cysteic acid residues 

 remain (after oxidation) in the residual material. The results of both experi- 

 ments at two levels of degradation, which are given in Table 8, indicate 

 that, within experimental error, there is excellent agreement between the 

 sum of the residues liberated and those recovered in the residual protein, 

 and with the previously reported^^ analysis of papain. It should be noted 

 that the absence of methionine or its sulfone from oxidized samples, and the 

 expected low recovery of histidine and phenylalanine are consonant with 

 the view that all the materials analyzed are derived from the mercuripapain 

 and not from the aminopeptidase. 



Specificity. One of the major reasons why the intensive study of papain 

 was undertaken is that a variety of synthetic substrates of known structure 

 can be studied. ^ It would appear from general experience that the specificity 

 of enzyme-substrate interaction can be more readily studied under these 

 circumstances than when the enzyme is limited in its action to one or a few 

 compounds. 



It was of great interest to study the relative specificity of papain at various 

 stages of degradation.^2-^^ The results with several synthetic substrates of 

 widely differing structure are given in Table 9 and the data obtained with 



Table 9 



SPECIFICITY OF DEGRADED PAPAIN 



A sample of Mg++-activated aminopeptidase (21 mg., Ci=60) was incubated at 40° and 

 pH 8-6 with 14-3 mg. of mercuripapain. Aliquots were removed at various intervals and 

 the extent of degradation was estimated by the ninhydrin method. Other aliquots were 

 tested with the substrates shown. Each test mixture contained 0-05 m substrate, 0-005 M 

 cysteine, 0-001 m ethylenediaminetetraacetate and 0-05 m acetate buffer at pH 5-2. Hydro- 

 lysis was estimated by titration with 0-01 n-KOH in 90 per cent ethanol. The Cx was 

 calculated on the basis of initial papain concentration. Optically active substrates were 

 of the L configuration. 



human serum albumin as substrate are shown in Fig. 6. These findings 

 demonstrate that, within experimental error, no change in substrate speci- 

 ficity occurs when as many as 70 of the 180 residues of papain are removed 



