11] STRUCTURE AND ACTIVITY OF PAPAIN 199 



from the enzyme. In essence, these results support the view, expressed ear- 

 ]jgj.^36-38 j-j^^j- substrate specificity and catalytic efiectiveness are part of the 

 same phenomenon, namely, that each locus of interaction on the enzyme, 

 which contributes to the specificity, plays a role in the catalytic transforma- 

 tion of the substrate. Perhaps this is just another way of stating that the 

 key to enzymic action resides in the formation of the enzyme-substrate 

 complex, a problem which will be also discussed below. 



40 



50 



10 20 30 



TIME (Minutes) 



Fig. 6. Action of papain and degraded papain (minus approximately 70 residues) on 

 human serum albumin tested at 40° and in 005 m dimercaptopropanol at pH 5-2 in 

 acetate buffer. (Hill and Smith, unpublished.) 



AUTODIGESTION OF PAPAIN 



Recent studies^^ have shown that crystalline mercuripapain or papain can 

 be readily chromatographed on IRC-50 in neutral or slightly acidic 0-2 m 

 phosphate buffers. At pH 7-0 the protein appears to be homogeneous, 

 whereas at pH 6-2 small amounts of material are found which appear in 

 advance of the main active component. On occasion, some of the faster 

 material showed some proteolytic activity, particularly when stored papain 

 was used for the chromatography. Inasmuch as we had known that stored 

 papain undergoes some autodigestion, a solution of active papain was 

 allowed to autolyze for a period. When this solution was chromatographed, 

 as shown in Fig. 7, it was found that the original active peak had diminished 

 in quantity and other, more rapidly eluted, fractions appeared. It is striking, 

 however, that a considerable portion of the proteolytic activity emerged 

 with the papain fragments well in advance of the peak of intact papain. 



The yield of active fragments is small, as might be anticipated, since 

 active papain can digest other molecules at a variety of sites. The afore- 

 mentioned results with the aminopeptidase digestion indicate that the active 

 region is not at the A^-terminal portion of the papain molecule. Hence it 



