264 INACTIVATION OF TRYPSIN. Ill 



It follows from this equation that the amount of free trypsin pres- 

 ent in a solution containing a given amount of inhibitor is a function 

 of the dilution. The more concentrated the solution the more trypsin 

 will be combined. Since the determination of the amount of free 

 trypsin was made by adding 1 cc. of the trypsin to 25 cc. of gelatin it 

 is possible for most of the trypsin to be active (uncombined) under 

 these conditions but nearly all combined (inactive and stable) in the 

 undiluted tr)rpsin solution. 



If the trjrpsin solution at the beginning of the experiment contains 

 undigested protein it is evident that the concentration of the inhibit- 

 ing substance, which is found by the action of trypsin on the protein, 

 will increase during the experiment, and that the observed result 

 will be the combination of two effects: first, reversible inactivation 

 of the tr3rpsin due to the presence of the inhibiting substance, and 

 second, irreversible destruction of the free trypsin. The combina- 

 tion of these two factors will give a curve which, compared to the curve 

 for the pure trypsin solution, will drop too rapidly at first, due to the 

 increasing concentration of inhibiting substance aiid will then de- 

 crease too slowly owing to the fact that the trypsin is nearly all com- 

 bined and therefore stable. This is the result shown in Fig. 1. This 

 explanation may be verified by diluting the solution sufficiently 

 before determining its activity. Since the inactivation due to the 

 inhibiting substance is reversible and depends on the concentration 

 of the solution whereas the spontaneous inactivation is irreversible, 

 the two effects may be separated in this way and the resulting value 

 alone will be a measure of the irreversible spontaneous decomposi- 

 tions. The result of an experiment performed in this way is shown 

 in Fig. 2. The curve for the formol titration of the trypsin solution 

 alone is also given. The figure shows that when the trypsin solution 

 is sufficiently diluted before the determination is made, the initial 

 rapid drop disappears and also that the time during which this drop 

 is noticeable in the less diluted solution corresponds to the time dur- 

 ing which the formol titration is increasing. 



There remains to be explained the subsequent retardation of the 

 inactivation in solutions containing protective substances. It follows 

 from the law of mass action which, as has been shown, correctly 

 expresses the equilibrium, that the smaller the amount of trypsin 



