160     Chemical  Reactions  of  Diphtheria  Antitoxin.  {  Am v^m' 
with  20  Cc.  of  n/I0°  NaOH,  and  after  10  minutes  was  neutralized 
with  20  Cc.  of  HQ  n/ioo.  This  solution  retained  the  full  anti- 
toxic value  of  the  original,  i.  e.,  short  contact  with  n/ioo  NaOH 
does  not  injure  antitoxin.  No  attempt  was  made  to  find  if  pro- 
longed contact  would  injure  it. 
Five  Cc.  of  globulin  I  were  treated  with  5  Cc.  NaOH  n/10,  then 
neutralized  with  5  Cc.  HC1  n/10.  This  was  diluted  to  150  Cc.  2 
Cc.  of  this  solution  protected  against  one  L  +  dose  of  the  toxin. 
Apparently  there  was  some  slight  loss  in  activity. 
Five  Cc.  globulin  II  was  diluted  with  5  Cc.  NaCl  (0.85  per  cent.), 
treated  with  10  Cc.  NaOH  n/10  and  neutralized  with  10  Cc.  HC1 
n/10.  This  had  about  the  full  antitoxic  value,  i.  e.,  n/20  NaOH 
does  not  injure  the  antitoxin,  at  least  in  this  preparation. 
Ten  Cc.  globulin  preparation  II  were  treated  with  10  Cc.  NaOH 
n/10  and  shaken  with  benzol.  The  benzol  residue  was  shaken  with 
25  Cc.  NaCl  (0.85  per  cent.)  Even  3  Cc.  did  not  protect  against  one 
L  -f-  dose  of  the  toxin.  The  mother  fluid  contained  the  full  antitoxic 
value,  showing  that  n/20  NaOH  did  not  injure  this  antitoxin,  and 
that  it  would  not  shake  into  alkaline  benzol. 
Ten  Cc.  of  globulin  II  were  treated  with  5  Cc.  NaOH  n/10  and 
shaken  with  ether.  The  ether  residue  contained  no  antitoxin,  while 
the  mother  fluid  had  its  full  value,  i.  e.,  antitoxin  is  not  soluble  in 
alkaline  ether. 
Five  Cc.  of  globulin  preparation  I  were  evaporated  to  dryness  in 
vacuo  and  extracted  twice  with  20  Cc.  of  methyl  alcohol  (Merck). 
This  alcohol  was  evaporated  and  the  residue  left  by  its  evaporation 
was  extracted  with  10  Cc.  normal  NaCl.  Even  3  Cc.  of  this  did  not 
protect  from  one  L  -f-  dose  of  the  toxin.  Presumably  no  antitoxin 
was  present. 
The  residue  after  methyl  extraction  was  dissolved  with  20  Cc. 
of  NaCl  (0.85  per  cent.)  and  10  Cc.  n/10  NaOH.  It  did  not  dis- 
solve in  NaCl.  It  was  then  neutralized  with  10  Cc.  n/10  HC1. 
This  solution  had  the  full  antitoxic  value  of  the  original  solution. 
The  diphtheria  antitoxin  as  present  in  this  preparation  is  insoluble 
in  methyl  alcohol. 
The  same  amount  of  preparation  II  was  evaporated  to  dryness 
and  treated  with  methyl  alcohol  containing  the  same  amount  of 
NaOH  as  used  in  the  above  test,  but  as  very  small  amounts  seemed 
to  be  dissolved,  no  tests  were  made  on  animals. 
Five  Cc.  of  preparation  II  were  treated  with  5  Cc.  of  n/10  NaOH 
