234 
Studies  on  Pepsin. 
Am.  Jour.  Pharm. 
April,  1919. 
to  be  free  from  phosphorus  and  to  contain  no  nucleoproteid,  but 
the  analyses  of  his  preparations  showed  no  constancy  in  results. 
That  a  protein-free  pepsin  solution  having  digestive  action  is 
possible,  has  also  been  maintained  by  Schrumpf.6  The  latter  pre- 
pared a  Buchner-pressed  extract  of  hog  stomachs,  clarified  by  filtra- 
tion, and  dialyzed  against  running  water.  The  dialysate  thus  ob- 
tained was  precipitated  by  addition  of  cholesterin  in  alcohol-ether 
solution,  filtered,  the  precipitate  redissolved  in  water,  and  the  sus*- 
pension  finally  clarified  by  a  Kitasato  candle.  The  clear  filtrate, 
while  giving  none  of  the  protein  reactions,  still  showed  powerful 
digestive  activity. 
The  amino  acid  constituents  of  pepsin  have  been  investigated  by 
Hugounenq  and  Morel'1'  using  an  autodigested,  hydrochloric  extract 
of  hog  stomachs.  They  conclude  that  an  extract  of  pepsin  contains 
a  number  of  monoamino  acids  in  the  free  state,  probably  formed  in 
the  autodigestion.  Glycocoll,  aspartic  and  glutaminic  acids,  and  also 
histidin,  they  found  to  be  absent  in  the  material  examined. 
It  is  thus  readily  apparent  that,  as  true  with  other  enzymes,  the 
chemical  nature  of  pepsin  is  still  an  open  question.  Nearly  all  of 
the  above  investigators  have  based  their  conclusions  on  crude  prep- 
arations, undoubtedly  containing  admixed  or  combined  impurities. 
Seemingly,  no  attempt  has  been  made  to  prove,  by  quantitative 
measurements  of  the  proteolytic  activity,  that  an  actual  purification 
has  taken  place,  where  such  is  mentioned.  The  present  investiga- 
tion was  undertaken  by  us  to  determine  what  changes  take  place  in 
the  purification  of  pepsin,  with  the  view  of  possibly  throwing  some 
light  on  the  chemical  nature  of  the  enzyme. 
Experimental  Procedure. 
Methods. — As  basic  material  for  purification,  a  composite  lot 
(consisting  of  a  number  of  different  samples)  of  1  : 2,000  commer- 
cial pepsin  was  employed.  Sufficient  stock  of  this  mixture  was 
reserved  to  enable  the  preparation  of  all  of  the  various  strengths  of 
the  enzyme  given  below.  The  weaker  samples  (up  to  1  : 18,000) 
were  prepared  by  fractional  precipitation  of  a  20  per  cent,  aqueous 
solution,  while  the  more  active  strengths  were  obtained  by  salting 
out  the  former,  filtering  and  dialyzing.    In  each  case,  the  final 
6  Schrumpf,  Beitr.  Holm.,  vol.  6,  396,  1905. 
7  Hugounenq  and  Morel,  Compt.  rend.,  vol.  147,  212,  1908. 
