184 EMIL L. SMITH, ROBERT L. HILL AND J. R. KTMMEL [11 



investigations^ show that the crystalline enzyme is a good antigen and the 

 system of protein and rabbit antiserum shows all of the behavior charac- 

 teristic of a single antigen and a single antibody. Moreover, since the anti- 

 serum strongly inhibits the enzymic activity, it is evident that the enzyme 

 itself is the antigen. In essence, all of these studies are consonant with the 

 view that crystalline papain is molecularly homogeneous. Some of the over- 

 all properties of the molecule are summarized in Table 1. 



THE SULFHYDRYL GROUP 

 AND SULFUR DISTRIBUTION 



Papain is inhibited by all of the agents which react with thiol groups, e.g., 

 heavy metal ions, organic mercurials, alkylating agents, and oxidizing agents. 

 The presence of an essential thiol group thus presents a number of disad- 

 vantages since the enzyme is readily inactivated by traces of metal ions and 

 by oxidation under aerobic conditions. Full enzymic activity is obtained 

 only when a chelating agent and a reducing agent are present. ^ Dimercapto- 

 propanol (BAL) serves in both capacities and is a convenient compound 

 to use in kinetic studies of the enzyme.*-^ 



The strong affinity of the enzyme for heavy metal ions has been useful 

 in permitting the isolation of crystalline mercuripapain which is inactive 

 and is thus not liable to autodigestion. ^ Moreover, mercuripapain is more 

 homogeneous and more stable than crystalline papain itself. Although crys- 

 talline mercuripapain contains one atom of mercury for each two moles 

 of protein, it has been possible to demonstrate that a molecule of reduced 

 papain can bind maximally one mercuric ion.^^^ 



The spectrophotometric method of Boyer^'' has been employed to esti- 

 mate the affinity of reduced papain for;j-chloromercuribenzoate (PCMB),^'^^ 

 Fig. 1 shows the results of such studies with different preparations of the 

 crystaHine enzyme. It is evident that in each case less than one thiol group 

 per mole of papain can react with the mercurial. In addition, the proteo- 

 lytic coefficient (Q) for the hydrolysis of benzoyl-L-argininamide, varies 

 with the amount of active SH. It should be noted that Balls and Lineweaver^^ 

 originally observed that different preparations of the crystaUine enzyme vary 

 in absolute activity. 



The finding that less than one mole of reactive SH is present in papain 

 does not indicate any contamination of the enzyme with other proteins. 

 On the contrary, crystalline preparations of the enzyme with different pro- 

 teolytic activity manifest the same physical properties, are homogeneous and 

 possess the same amino acid composition. The explanation for the varia- 

 tion in activity and reactive thiol resides in the observations that preparations 

 of the enzyme or its mercury derivative undergo an irreversible inactivation 

 on storage. Indeed, this change appears to occur in the crude latex as well, 

 and freshly prepared samples of crystalline enzyme manifest this variability.^ 



