VOL. 12 (1953) MECHANISM OF ENZYME ACTION. LV 65 



cobalt, magnesium and barium, enumerated in order of decreasing effect, are also 

 reported as slightly increasing the activity of the enzyme^^. Whereas the first three ions 

 are about equivalent in their effect, the second group of ions exerts a much smaller 

 effect. On electrophoresis the ions of the second group, even at higher concentrations, 

 do not give rise to the separation of the moving boundary into its two components. 

 Instead, a single boundary is obtained. The binding of these ions, resulting in an increased 

 activity of the enzyme, is apparently more loose than that of the first group of ions, 

 and falls therefore below the threshold value apparently necessary to prevent the inter- 

 action of enzj-me molecules resulting in the formation of polymeric forms. 



It was, of course, uncertain whether both of the components are actually trypsins 

 or whether one is an inactive impurity or product of a denaturation of the enzyme. The 

 results of the direct assay of the two components, reported in Table III, clearly show that 

 they possess comparable proteolytic activity. Furthermore, experiments depicted in 

 Fig. 2 and Table IV indicate that both components show closely similar stabilities, 

 namely they are self-digested at about the same rate, either in presence or absence of 

 calcium. It is obvious, therefore, that calcium offers an equally protective effect to both 

 components and that we may therefore consider that both are trypsins. This would be 

 in agreement with the behaviour of another example of a multi-component protein all 

 fractions of which have the same biological activity, namely the antitryptic factor of 

 eggwhite, ovomucoid^. 



ACKNOWLEDGEMENT 



This study was carried out under the aegis of the U.S. Atomic Energy Commission. — We also 

 wish to record the assistance of L. Terminiello. 



SUMMARY 



1. In the neighbourhood of pH 5 the electrophoretic analysis of crystalline trypsin results in 

 patterns characteristic of a homogeneous protein, while the addition of calcium, manganese or calcium 

 ions (0.033 ^■^) causes the moving boundary to separate into two distinct peaks. The two components 

 of trypsin have comparable stability both in the presence or absence of calcium ions and, if separated 

 electrophoretically, possess similar proteolytic activity. 



2. Trypsin has a sedimentation constant of 2.4-2.5 5 at the pH values of 2.3 and 8. On the acid 

 side of pH 2.3, the denaturation causes a slow appearance of faster sedimenting components, whereas 

 at pH 8, the self-digestion prompts the appearance of slower sedimenting breakdown products. The 

 rate of sedimentation of the remaining unaltered trypsin appears unaffected by the presence of either 

 the faster or the slower sedimenting components. 



3. In the intermediate pH range, around pH 5, the sedimentation constant may increase up to 

 3.5 S, depending upon the conditions, while the enzyme sediments as a homogeneous protein. This 

 is due to a reversible aggregation equilibrium which is strongly temperature dependent. Calcium 

 prevents the aggregation. 



4. Trypsin has to be viewed as a heterogeneous protein consisting of two components. In the 

 absence of calcium, manganese or cadmium, in the neighbourhood of pH 5, the reversible molecular 

 interaction of the two components induces the aggregation and results in apparently homogeneous 

 electrophoretic and ultracentrifugal patterns. The addition of calcium or of the other two ions 

 prevents the interaction, restores the normal sedimentation behaviour, and reveals the true electro- 

 phoretic heterogeneity of the enzyme. 



RfiSUME 



I. Au voisinage de pH 5, la trypsine cristallisee se comporte, a I'electrophorese, comme une pro- 

 teine homogene, tandis qu'apres addition des ions de calcium, manganese ou cadmium (0.033 M), 

 elle se separe en deux constituants distincts. Les deux constituants ont une stabilite comparable 

 a la fois en presence et en absence des ions de calcium et, apres separation par electrophorese, pos- 

 sedent des activites proteolytiques semblables. 



References p. 66. 



