11] STRUCTURE AND ACTIVITY OF PAPAIN 183 



Other proteolytic enzymes, synthetic substrates of known structure can be 

 readily prepared. Because of the good stability of the enzyme under a variety 

 of conditions, kinetic studies with substrates of different structure can be 

 performed and such studies have yielded valuable information concerning 

 the nature of the active site and a possible mechanism of action of the 

 enzyme.^-* 



One of the most important features of papain is the finding that a single 

 sulfhydryl group is essential for activity and that this group is intimately 

 concerned in the enzymic mechanism.^-^ This presents the opportunity of 

 labeling the specific part of the peptide chain which is directly involved in 

 the interaction with substrates. 



This brief consideration of some of the desirable properties of the enzyme 

 should not serve to obscure the fact that a protein containing approxi- 

 mately 1 80 residues poses difficult problems for study. Although satisfactory 

 methods appear to be available for determining the amino acid sequence, 

 it is obvious to all who are engaged in such studies that the technical prob- 

 lems are formidable indeed and that the labor involved is considerable. 



In this presentation we shall discuss some of the studies on crystalline 

 papain which indicate the approaches which are being used in our labora- 

 tory. Major emphasis will be given to studies of the overall structure and of 

 attempts to determine the nature of the active region of the molecule by 

 enzymic degradation of the molecule, by labeling the active group, and by 

 kinetic analysis. 



GENERAL PROPERTIES AND HOMOGENEITY 



Papain has a molecular weight of about 20,500 as estimated by both physical 

 and chemical methods. (Table 1.) The molecule is slightly basic with an iso- 



Table 1 

 SOME PROPERTIES OF PAPAIN 



electric point at pH 8-75 in buffers of 0-1 ionic strength. The crystalline 

 enzyme, or better, its crystalhne mercury derivative, is essentially homo- 

 geneous as judged by sedimentation studies in the ultracentrifuge/ by elec- 

 trophoretic analysis,^ and by end-group determinations.' Immunochemical 



