256 
Studies  on  Pepsin. 
5  Am.  Jour.  Pharm. 
I     April,  1921. 
EXPERIMENTAL  PROCEDURE. 
Methods. — As  basic  material  for  purification,  a  composite  lot 
(consisting  of  a  number  of  different  samples)  of  1  :2coo  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  :  18000) 
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 
purified  material  was  dried  to  a  constant  moisture  content  of  about 
5  per  cent,  and  scaled.  Assays  for  proteolytic  power  were  then 
carried  through  and  the  samples  analyzed  chemically. 
Determination  of  the  proteolytic  strength  of  the  different  sam- 
ples, made  in.  association  with  our  colleagues,  L.  M.  Gerdes  and 
W.  L.  Seibert,  was  in  accordance  with  the  method  given  in  the  9th 
revision  of  the  U.  S.  Pharmacopoeia.  The  assays  were  checked  in 
each  case,  and  controlled  by  running  through  a  standard  (1  :  3000) 
pepsin  under  identical  conditions. 
The  chemical  examination  included  analyses  of  total  mineral 
matter,  total  nitrogen,  toal  sulphur  by  the  method  of  Wolf  and 
Osterberg,  volumetric  estimation,  in  the  ash,  of  phosphoric  acid 
as  P2O5,  chlorides  as  NaCl,  calcium  as  CaC03 ;  also,  determination 
of  nitrogen  existing  in  coagulable  protein,  proteoses  by  zinc  pre- 
cipitation, peptones  by  Bigelow  and  Cook's  modification  of  Sjern- 
ing's  method,  and  amino  acids  according  to  Van  Slyke.  In  addi- 
tion, observations  were  made  in  a  2  per  cent,  aqueous  solution  of 
optical  rotation,  and  of  the  hydrogen-ion  concentration.  The  direct 
reading  ionometer  described  by  Bartell  was  used  in  the  latter,  with  a 
Weston  Standard  Cell,  and  the  chain:  Calomel  electrode  (A/KCl)  — 
saturated  KC1 — pepsin  solution — Pt  electrode — H2  at  23  degrees. 
The  complete  "set  up"  employed  was  similar  to  that  used  by  Davis 
in  a  previous  investigation  of  diphtheria  toxin. 
Supplementing  the  preceding,  qualitative  tests  were  carried 
out  in  accordance  with  the  technique  employed  by  one  of  us,  Davis, 
with  peptone  samples.  Both  a  straight  2  per  cent,  aqueous  solution 
and  the  filtrate,  after  coagulating  the  protein,  were  used,  and  exam- 
ination made  for:  tyrosin  (xanthoproteic,  Millon's  reaction)  trypto- 
phane (Adamkiewicz  Hopkins-Cole  reagent),  glycoprotein  and 
glycoproteoste  (Molicch  reagent).  Tests  were  also  made  on  the 
filtrate  from  coagulable  protein,  for  proteoses  (by  addition  of  satu- 
