400 Dr. J. Mellanby. ) [June 3, 
Brodie (3) found that the same result could be obtained by half saturation 
with ammonium sulphate. 
Belfanti and Carboni confirmed the results of Brodie and Dieudonné. 
Freund and Sternberg (4) attempted the isolation of diphtheria antitoxin 
from serum by using 1°6-per-cent. potash alum. They stated that the filtrate 
obtained from serum after adding this salt, when treated with ammonium 
sulphate, gave a precipitate which contained the greater part of the antitoxin 
associated with only a small quantity of the original protein of serum. 
Pick (5) attempted to differentiate the antitoxic portion of serum by 
fractional precipitation with ammonium sulphate. 
Préscher first digested antitoxic serum with trypsin and then fractionated 
the resultant liquid by means of ammonium sulphate. He stated that in this. 
way he obtained a solution containing all the antitoxin free from protein. 
THE RELATION OF DIPHTHERIA ANTITOXIN TO THE NORMAL PROTEINS OF 
HorsE SERUM.* 
Three proteins possessing well-defined physical properties may be separated 
from horse serum. These proteins are present in serum in the following 
quantities :— 
(@)e Globulin eeeeee, oe About 3 per cent. 
(0)? Alum (a) BS Oa es 
Kc). “Albumin (3) ene Faint PaaS 
In the investigation of the properties of diphtheria antitoxin the first 
question to settle was whether it belonged to the protein groups (a), (0), 
Onc): 
The protein (a)—globulin—was obtained by precipitating antitoxic serum 
with 10 volumes of water and neutralising with acetic acid. The precipitate 
was adequately washed by decantation and filtration. This precipitate when 
dissolved in sodium chloride did not possess antitoxic properties. It may 
therefore be concluded that diphtheria antitoxin is not a globulin as rigidly 
defined. This result confirms the work of Dieudonné and Belfanti and 
Carboni. 
The protein (c)—albumin (8)—is probably that protein of serum which can 
be crystallised by means of ammonium sulphate and sulphuric acid. 
Crystalline albumin was prepared from antitoxic horse serum by this method. 
* The experiments detailed in the following pages are based upon results previously 
described in two papers: “The Physical Properties of Horse Serum,” ‘ Journ. Physiol.,’ 
vol. 35, p. 473, 1907, and “The Precipitation of the Proteins of Horse Serum,” ‘Journ. 
Physiol.,’ vol. 36, p. 288, 1907. 
