406 Dr. J. Mellanby. [June 3, 
The Action of Ferments on Diphtheria Antitoxin. 
0-2 per cent. hydrochloric acid has been stated above to have no 
destructive action on diphtheria antitoxin. But when serum is acidified 
with 0:2 per cent. hydrochloric acid and a quantity of pepsin is added, it is 
found that the antitoxic value diminishes at a rapid rate. This destruction 
of diphtheria antitoxin by pepsin in vitro is in line with the inability to 
immunise animals against a subcutaneous injection of diphtheria toxin by 
feeding with antitoxic serum. Trypsin also destroys diphtheria antitoxin, 
but much more slowly than pepsin. 
With regard to the statement of Préscher concerning the preparation of 
protein-free antitoxin by digesting antitoxic serum with trypsin, the following 
experiment is of interest. 
400 cc. of trypsin (Liquor pancreaticus Benger) were added to 2000 c.c. 
of serum and incubated at 37° C. After four and nine days respectively the 
quantitative precipitation of this digested mixture by ammonium sulphate was 
determined. The following results were obtained :— 
After four days the amount of solid not precipitated by saturation with 
ammonium sulphate was 10 per cent. of the total solid; after nine days the 
quantity of solid not precipitated had increased to 24 per cent. of the total 
solid. The 14 per cent. increase represented digested protein. 
The quantitative results for the precipitation of the proteins of serum after 
four and nine days’ tryptic digestion were determined. 
Percentage of protein not precipitated. 
Percentage of Am,SO, (solid). 
After 4 days, After 9 days. 
18 °75 82 °4 98 :2 
20°0 74°5 91°6 
21°25 _ 17 0 
22 °5 55 °8 70 °0 
25 ‘0 42-0 56 °2 
27°5 34:0 46 °6 
30 ‘0 26 ‘6 40 °7 
35 °O 20 ‘0 — 
— 31-7 
| 40°0 
There are two results to be noted from these figures :— 
(1) The action of trypsin on the proteins of serum is slow, but progressive. 
(2) The diminution of the amount of protein precipitated by ammonium 
sulphate after tryptic digestion is constant for all quantities of ammonium 
sulphate. Thus, in the above figures the average difference between the 
amount of protein left in solution by any amount of ammonium sulphate 
