232 INACTIVATION OF TRYPSIN. I 



before adding the formaldehyde. The solution was then titrated to 8.2 with 

 thymol blue. The titrations were made with 0.1 N NaOH. The end-points 

 are both accurate to 0.05 cc. 



Technique of the Determination. — The gelatin solution is melted, 25 cc. pipetted 

 into a series of the conductivity cells and the cells suspended in the water bath 

 at 33° ± 0.01°C. The conductivity is determined at intervals until it becomes 

 constant (usually about 20 minutes). 1 cc. of the trypsin solution (previously 

 warmed to 33° and having the same conductivity) is then added. The solution 

 is then thoroughly mixed by sucking back and forth three times in a warm dry 

 15 cc. pipette. It is necessary to avoid air bubbles and to be sure that the solution 

 is well mixed. Irregular results can nearly always be traced to incomplete mixing. 

 The conductivity of the solution is then read at intervals so that readings are 

 obtained at every 1.5 or 2 divisions on the bridge, until the reading is 485 or less 

 (corresponding to a decrease of 15 points). The elapsed time is calculated from 

 the time at which the trj^jsin is added. Since in order to obtain the elapsed time 

 it is necessary to make a great many subtractions it is a great convenience to use 

 a clock which is divided into hundredths of an hour instead of minutes. If the 

 trypsin solution has been carefully adjusted to the same temperature and con- 

 ductivity as the gelatin and the mixing carried out without change of temperature 

 or the formation of air bubbles, it will be found that the readings form a perfectly 

 smooth curve. It frequently happens, however, that the first reading (before 

 the trypsin is added) does not fall on the same curve as the subsequent readings. 

 In this case the curve is extrapolated back from the first reading after the trypsin 

 is added in order to find the zero reading. Since, as was stated above, the curve 

 is perfectly smooth when the experiment is done with sufficient care this pro- 

 cedure seems justified. The results should always fall on a smooth curve after 

 the first 0.05 hour (corresponding to a change of from 0.1 to 1.0 on the bridge). 

 The results are then plotted on a large scale and the time necessary for the reading 

 to change 10 points determined by interpolation. This corresponds to an actual 

 change in the conductivity of 0.0782 X 10~^ reciprocal ohms and is less than 

 10 per cent of the total change which can be effected by the trypsin under these 

 conditions. Owing to the large number of experiments, the individual time 

 curves from which the time necessary to cause the 10 points change is determined 

 will not be given, but only the time interval interpolated from these curves. The 

 points lie so close together on the curve that there is little or no possibility of 

 arbitrary adjustment of the curve. The elapsed time is, therefore, a direct 

 experimental determination. In the few cases where it was possible to draw 

 more than one curve through the experimental points, the extreme values for the 

 interpolated time have been given. 



