Separation of Antitoxin from its Associated Proteins. 9 



series of the same antitoxic serum was heated for from 6 to 72 

 hours in closed containers, at a temperature of 57° C. After cool- 

 ing to room temperature, the series was saturated with sodium 

 chloride and brought up to a dilution of i : 10 with saturated 

 sodium chloride solution. Twelve hours later the resulting pre- 

 cipitations were filtered off. Potency tests on these filtrates showed 

 a loss of 5 per cent, after heating 6 hours and an increasing loss 

 up to 22 per cent, after heating 72 hours. The protein converted 

 into an insoluble condition (in saturated sodium chloride solution) 

 was 30 per cent, for the 6-hour period, increasing up to 48 per 

 cent, for the 72-hour period. The increase of antitoxic units, per 

 gram protein, was 3 5 per cent, after 6 hours heating, increasing 

 up to 53 per cent, after 48 hours. 



Owing to the larger per cent, destruction of antitoxin at the 72- 

 hour heating than the per cent, increase on conversion, the potency 

 per gram protein dropped to 52 per cent, increase over the native 

 serum. On separating the remaining unconverted albumin from 

 this series, the increase of antitoxic units, per gram protein, was 

 60 per cent, after 6 hours heating, increasing to 78 per cent, 

 after 48 hours. The 72-hour heating showed an increase of 73 

 per cent, over the native serum. 



Citrated plasma under the same conditions gave practically the 

 same results. Gibson's antitoxic globulin solution (blood alka- 

 linity) containing only that globulin soluble in saturated sodium 

 chloride solution was heated under the same conditions. The 

 potency loss was 5 per cent, for the 6-hour period, and an increas- 

 ing loss up to 23 per cent, for the 72-hour period. The soluble 

 globulin converted into an insoluble condition (in saturated sodium 

 chloride solution) was 30 per cent, after 6 hours' heating, in- 

 creasing to 47 per cent, after 72 hours. The increase of anti- 

 toxic units, per gram of protein, was 37 per cent, after 6 hours' heat- 

 ing, increasing to 54 per cent, for the 24 hours. Here again 

 the 72-hour heating period caused a larger per cent, destruction 

 of antitoxin than per cent, globulin converted into an insoluble 

 condition (in saturated sodium chloride solution), dropping to an in- 

 crease of 46 per cent., per gram protein, over Gibson's antitoxin 

 globulin solution. This work which is being carried out further is 

 practically and scientifically important, and may throw some light 

 on the chemical characteristics and the nature of antitoxins. 



