174 
Note  on  Trypsin. 
Am.  Tour.  Pharm. 
March,  1919. 
cipitated  by  safranine  is  actually  trypsin,  since  the  supernatant 
liquid,  although  containing  substances  precipitated  by  alcohol,  is 
practically  free  from  enzymes.  Holzberg  has  repeated  Robertson's 
experiment.  To  solutions  of  Griibler's  trypsin,  Fairchild's  trypsin, 
and  aqueous  extracts  of  sheep's  pancreas,  he  added  three  eighths  of 
the  volume  of  a  0.8  per  cent,  solution  of  safranine.  The  precipitates 
were  collected  and  washed,  and  their  proteolytic  action  tested  by 
digesting  with  a  solution  containing  2  Gms.  of  casein  (Gross  test). 
The  results  are  given  in  the  following  summary:  The  substance 
which  is  precipitated  by  the  addition  of  safranine  to  aqueous  solu- 
tions of  Griibler's  or  Fairchild's  trypsin,  or  to  aqueous  extracts  of 
pancreas,  has  a  strong  proteolytic  action,  contains  safranine,  is  very 
sparingly  soluble  in  water,  and  insoluble  in  organic  solvents.  After 
the  substance  has  been  removed  from  aqueous  solutions  of  com- 
mercial trypsin  or  pancreas  extract,  a  considerable  quantity  of  a 
substance  remains  in  solution,  which  is  precipitable  by  alcohol,  and 
in  the  case  of  pancreas  extract,  a  further  precipitate  is  obtained  by 
means  of  an  alcohol-ether  mixture  (three  to  one).  These  sub- 
stances are  practically  devoid  of  preteolytic  activity. 
I  repeated  this  experiment  by  adding  a  0.1  per  cent,  and  also  a 
saturated  solution  of  safranine  to  various  infusions  containing  tryp- 
sin. An  aqueous  extract  of  the  pancreas  gave  a  considerable  pre- 
cipitate, which  behaved  in  exactly  the  same  way  as  the  precipitate 
obtained  by  Holzberg.  Instead  of  using  the  Gross  test,  I  used  the 
gelatin  test.4  I  found  it  impossible  to  separate  the  precipitate  from 
the  filter,  as  it  dried  into  a  horny  film.  The  paper  was  therefore 
cut  up  into  small  pieces,  and  in  order  to  obtain  some  quantitative 
data  it  was  cut  into  one-inch  squares,  which  were  weighed,  and 
these  were  then  ruled  into  100  divisions  and  the  small  squares  placed 
in  the  gelatin  tubes.  It  was  found  that  six  squares  liquefied,  but 
five  did  not.  The  approximate  weight  of  precipitate  on  one  square 
inch,  assuming  it  to  be  uniform,  was  5.5  mgrms.  The  precipitate 
on  0.01  sq.  in.  would  therefore  be  0.055  mgrms.,  so  that  the  quantity 
of  dry  precipitate  which  is  active  lies  between  0.33  and  0.27  mgrm. 
Since  the  whole  of  the  gelatin  was  liquefied,  it  was  evident  that 
the  substance  causing  the  liquefaction  was  soluble,  and  it  therefore 
seems  that  Holzberg,  by  washing  his  precipitate,  has  removed  con- 
siderable quantities  of  enzyme.  On  repeating  the  experiments  with 
one  of  my  own  trypsin  preparations,  which  was  comparatively  free 
4  Journal  of  Society  of  Chemical  Industry,  1912,  1105. 
