Ion?:  pharm-  }  Chemical  Reactions  of  Diphtheria  Antitoxin.  161 
April,  1917* 
and  40  Cc.  methyl  alcohol  (Merck),  and  after  standing  about  20  to 
30  minutes  were  centrifugalized.  The  precipitate  was  dissolved 
in  25  Cc.  n/100  NaOH  and  then  neutralized  with  the  same  amount 
of  n/100  HQ.  This  gave  a  solution  much  the  color  of  the  original 
globulin  preparation,  although  the  precipitate  was  only  slightly  col- 
ored. This  solution  was  slightly  less  active  than  the  original  prep- 
aration, perhaps  due  to  standing,  as  several  days  elapsed  before  we 
were  able  to  test  this  preparation. 
10  Cc.  of  globulin  preparation  I  were  diluted  with  10  Cc.  of  NaCl 
(0.85  per  cent.)  and  precipitated  with  a  solution  of  lead  subacetate, 
drop  by  drop,  from  a  burette.  The  preparation  was  then  centrif- 
ugalized and  the  supernatant  fluid  precipitated  with  sodium  hydrogen 
phosphate,  centrifugalized  and  then  diluted  to  150  Cc.  Even  3 
Cc.  did  not  protect  against  one  L  -f-  dose  of  the  toxin. 
Several  other  attempts  were  made  to  free  antitoxin  from  pro- 
teins by  means  of  lead  subacetate  solution,  but  in  most  cases  the  fil- 
trate when  freed  from  lead  was  inactive.  In  one  case  it  possessed 
slight  antitoxic  value,  but  in  this  case  a  possible  excess  of  the  alka- 
line lead  solution  may  explain  the  result,  i.  e.,  solubility  of  antitoxin 
or  globulin  in  weak  alkali.  In  this  latter  case  the  lead  filtrate  con- 
tained a  few  antitoxic  units,  yet  produced  no  anaphylactic  reaction 
in  a  guinea  pig. 
10  Cc.  of  globulin  preparation  I  were  diluted  with  10  Cc.  of 
sodium  chloride  (0.85  per  cent.)  and  precipitated  with  a  cold  sat- 
urated aqueous  solution  of  picrolonic  acid  by  means  of  a  burette, 
then  centrifugalized.  The  centrifugalized  fluid  was  shaken  with 
acetic  ether  to  remove  picrolonic  acid,  at  least  partly,  although  acetic 
ether  did  not  seem  to  us  as  suitable  for  this  purpose  as  isobutyl 
alcohol.  After  separating  the  undissolved  acetic  ether,  the  solution 
was  diluted  to  an  arbitrary  amount  (250  Cc.)  Even  3  Cc.  of  this 
solution  failed  to  protect  against  one  L  +  dose  of  the  toxin.  The 
precipitate  was  shaken  with  NaCl  .85  per  cent,  and  made  into  a  col- 
loidal suspension  of  250  Cc.  Only  1  to  3  Cc.  were  tested.  1  Cc.  of 
this  suspension  protected  against  one  L+  dose  of  the  toxin,  show- 
ing that  most  and  perhaps  all  of  the  antitoxin  was  in  the  picrolonic 
acid  precipitate. 
A  similar  preparation  was  also  precipitated  with  picrolonic  acid. 
The  precipitate  was  shaken  into  a  colloidal  solution  or  suspension 
with  distilled  water.  This  was  centrifugalized  and  the  sediment 
shaken  with  NaCl  (0.85  per  cent.).    The  H20  solution  was  diluted 
S 
