December,  lii™' }  Standardization  of  Commercial  Papain.  545 
basis  of  the  quantitative  formation  of  carboxyl  groups  when  casein  is 
digested  in  slightly  alkaline  solution.  R.  Delaumay  and  O.  Bailly  7 
state  that  papain  is  a  peptone- forming  enzyme.  They  find  that  there 
is  no  relation  between  the  proteolytic  and  milk-coagulation  powers 
of  papain.  They  recommend  that  the  method  of  assay  be  based  on 
the  amount  of  protein  dissolved  in  unit  time. 
The  work  which  has  been  done  in  this  laboratory  for  the  pur- 
pose of  standardization  is  based  upon  the  work  of  Mendel  and 
Blood,8  and  a  number  of  commercial  samples  have  been  examined 
with  the  methods  there  given.  We  will  therefore  outline  the  methods 
used,  tabulate  our  results,  and,  lastly,  state  our  conclusions  at  the 
end  of  the  paper. 
Experimental. 
The  methods  other  than  those  used  in  studying  the  proteolytic 
activity  were  those  of  the  Official  Agricultural  Chemists.9  The 
directions  for  the  protein  digestions  were  as  follows : 
Preparation  of  Solutions. —  (a)  Egg-white  solution.  Separate 
the  whites  of  six  freshly-laid  eggs  and,  after  beating  slightly,  dilute 
with  two  volumes  of  1  per  cent,  sodium  chloride  solution.  Mix. 
Filter  through  plaited  filter  paper.  Make  up  to  a  definite  volume  10 
and  mix  thoroughly. 
(b)  Weigh  one  gramme  of  the  papain  and  transfer  to  a  dry 
100  c.c.  graduated  flask.  Do  not  make  up  the  solution  until  every- 
thing is  ready  for  the  determination.  The  papain  is  then  taken  up 
with  1  per  cent,  salt  solution,  shaken  thoroughly,  and  made  exactly 
to  volume.  Exactly  30  minutes  should  elapse  from  the  time  the  salt 
solution  is  poured  upon  the  papain  until  the  aliquots  of  the  solution 
are  taken. 
(c)  N/2  acetic  acid. 
Determination  of  Proteolytic  Activity  at  8o°  to  ioo°  C. — Into  a 
clean,  dry  50  c.c.  Erlenmeyer  flask  place  15  c.c.  of  standardized  egg- 
white  solution,  add  1  c.c.  of  the  papain  solution  and  then  9  c.c.  of 
1  per  cent,  salt  solution.  All  the  digestions  are  made  in  a  volume  of 
25  c.c.    Transfer  at  once  to  the  thermostat,  already  regulated  at 
7  Bull.  Sci.  Pharmacology,  20,  141-7  (1913). 
8  Jour.  Biol.  Chem.,  viii,  177  (1910). 
0  U.  S.  Dept.  Agr.  Bur.  of  Chem.  Bull.  107  (revised). 
10  This  solution  should  be  so  made  that  15  c.c.  contains  0.4000  gramme 
coagulable  protein.  This  requires  a  preliminary  determination  and  subse- 
quent dilution. 
