VOL. 12 (1953) 



MECHANISM OF ENZYME ACTION. LV 



63 



DISCUSSION 



The properties of the trypsin molecule studied in the present series of experiments 

 are summarized in Fig. 5 in which the behaviour of the enzyme at different pH ranges 

 is presented schematically. 



Maximum stabilit}^ 

 pH 2.3 



Denaturation 

 (Monomolecular) 



5 = 2.4 

 Heavier 



Components 



Molecular interaction: 



Temperature dependent aggregation 

 Electrophoresis: one component 



Calcium: 



Isoelectric point 

 pH 10.8 



Proteolysis 

 Equilibrium native ^ denaturated 



Calcium 

 < 



Selfdigestion (Bimolecular) 



Calcium protects 



s = 2.5 



Breakdown products 



Lower H"* 



binding 



No aggregation 



Electrophoresis: two components 



II pH 



Fig. 



The enzyme is most stable at about pH 2.3 and presents an ultracentrifugal beha- 

 viour characteristic of a homogeneous protein with a sedimentation constant s^Qyw = 2.4 

 S. At lower pH values the enzyme becomes increasingly unstable and the denatu- 

 ration causes the appearance of faster sedimenting aggregation products. The proteo- 

 lytic activity of the enzyme is exerted between pH 6 and 9 and in this pH range it is 

 known that its native and denaturated forms are in equilibrium^". Due to the digestion of 

 the latter by the native enzyme, its solutions rapidly lose their activity and slower sedimen- 

 ting breakdown products appear. Calcium and manganese ions when present reduce the 

 rate of self-digestion considerably and also slightly increase the activity of the enzyme. 

 It is assumed that these ions are integral parts of a more stable trypsin molecule, i.e. 

 that they cause a shift in the equilibrium between the native and denaturated enzyme 

 towards the active form. Taking advantage of the protective effect of calcium ions it was 

 possible also to establish that the iso-electric point of the enzyme is in the neighborhood 

 of pH 10.86. 



It is between the above two extreme pH ranges that a number of other interesting 

 phenomena occur. The formation of a specific calcium-trypsin complex was proven bj' 

 the study of the electrometric titration curves of the enzyme. In the pH range 2 to 5 

 where the dissociation of the carboxyl groups of the enzyme takes place, calcium ions, 

 in contrast to magnesium, sodium or potassium ions, induce a shift in the dissociation 

 curves towards the more acid pH values. This is evidence of a specific binding of the 

 calcium ions by the carboxyl groups of the enzyme which results in a decrease of their 

 dissociation constants. In this pH range, therefore, the net charge of the enzyme is 

 reduced by the addition of calcium ions. It would follow therefrom that its electrophoretic 

 mobility should be lower when calcium ions are added as compared to magnesium. 

 Experiments have shown, however, that the effect of calcium ions is more complex. 

 Whereas in the absence of these ions the enzyme gives rise to an electrophoretic pattern 

 characteristic of a homogeneous protein, the presence of calcium induces the separation 



References p. 66. 



