VOL. 12 (1953) 



MECHANISM OF ENZYME ACTION. LV 



59 



TABLE II 



ELECTROPHORETIC BEHAVIOUR OF TRYPSIN IN PRESENCE OF VARIOUS IONS 



* The ratio of areas of the two ascending boundaries was calculated as 2.3 ± 0.3, in 10 meas- 

 urements carried out under varying conditions. 



high concentrations of trypsin required in these analyses (0.5-1%) only a few drops of 

 each of the two components are required for the determination of their proteolytic 

 activity, carried out at a hundredfold dilution. By using a larger cell (6 ml) and a very 

 slowflowof buffer into the electrode vessel on the ascending side arm of the electrophoresis 

 cell, the separation of the two components could be extended to 24 hours at the end of 

 which period small samples of the two components became available using a syringe. 

 In Table III the relative activities of the components obtained in four independent 



TABLE III 



COMPARISON OF THE PROTEOLYTIC ACTIVITIES OF THE TWO COMPONENTS OF CRYSTALLINE TRYPSIN 



Slower component 



Faster component 



Experiment 



Opt. Dens. 

 of sample 



Activity 



Opt. Dens." 

 of sample 



Activity 



* Optical density at 280 m^ of the electrophoretically separated samples, diluted to 4 ml. 

 ** Activity expressed in arbitrary units (optical density at 280 m/< of hemoglobin digests after 

 hundredfold dilution of the separated samples). 



experiments, together with the protein concentration in each as measured by the ultra- 

 violet adsorption at 280 m^, are reported. Whereas no exact comparison of the specific 

 activities of the two components can be made on the basis of these experiments, there 

 is no doubt that both possess proteolytic activity. 



There was another opportunity to compare the properties of the two components of 

 trypsin, namely by subjecting trypsin solutions to selfdigestion at pH 8 in the presence or 

 absence of calcium. This ion, of course, considerably diminishes the rate of self-digestion. 

 Aliquot samples from the two solutions were taken at 24 hour intervals and examined 

 electrophoretically after dialysis with calcium buffer at pH 5 under conditions giving 

 the best separation of the two components. At the same time the tryptic activity was 

 determined. Due to dialysis at pH 5 most breakdown products were eliminated and a 



References p. 66. 



