546  Standardisation  of  Commercial  Papain.  {ADeoSrPwS1' 
80 °  C,  and  allow  the  digestion  to  proceed  for  exactly  15  minutes. 
Now  add  1  ex.  of  N/2  acetic  acid  and  transfer  immediately  to  a 
bath  at  ioo°  C.  and  heat  for  ten  minutes.  The  time  factor  should  be 
given  the  sharpest  attention. 
Bath  at  8o°  C. — 15  minutes. 
Transfer — 1  minute. 
Bath  at  ioo°  C. — 10  minutes. 
In  order  to  faciliate  the  acidification,  a  two-holed  stopper  is  used, 
bearing  a  long  glass  tube  to  serve  as  a  condenser,  and  a  small 
funnel  into  which  1  c.c.  of  acetic  acid  can  be  easily  placed. 
The  undigested  protein  is  filtered  off  on  a  tared  filter  paper. 
Wash  free  from  chlorides.  Wash  with  10  c.c.  of  95  per  cent, 
alcohol,  and  when  this  has  passed  through  add  10  c.c.  of  ether  U. 
S.  P.    Dry  at  ioo°  to  105 0  to  constant  weight. 
At  the  same  time  that  the  above  digestion  is  carried  out,  the 
amount  of  protein  in  the  egg-white  solution  coagulable  by  heat  is 
determined  in  a  blank,  i.e.,  15  c.c.  of  the  same  egg-white  solution 
is  mixed  with  10  c.c.  salt  solution  (or,  better,  9  c.c.  salt  solution  and 
1  c.c.  of  the  papain  solution  in  which  the  enzyme  has  been  destroyed 
by  boiling  vigorously  for  15  minutes)  and  the  operations  are  carried 
out  upon  this  mixture  exactly  as  described  above. 
Calculate  the  percentage  of  protein  rendered  non-coagulable  under 
these  conditions. 
Test  for  Pepsin  in  Papain. — Take  15  c.c.  of  the  same  egg-white 
solution  as  prepared  for  the  first  digestion.  Add  2  c.c.  1  per  cent, 
salt  solution,  3  c.c.11  of  N/2  HQ  and,  lastly,  5  c.c.  of  a  1  per  cent, 
papain  solution.  Add  0.5  c.c.  toluol  to  prevent  putrefaction.  Digest 
at  400  C.  for  15  hours.  Add  25  c.c.  of  a  10  per  cent,  solution  of 
trichloracetic  acid.  Heat  to  boiling  on  an  electric  stove.  Boil  ten 
minutes  and  filter  through  a  tared  paper,  and  wash  the  coagulum 
free  from  acid.  Wash  with  alcohol  and  ether.  Dry  at  ioo°  to 
1050  C.  to  constant  weight.  At  the  same  time  that  this  digestion  is 
carried  out  the  total  amount  of  coagulable  protein  present  should 
be  determined  in  a  blank  experiment. 
11  For  the  determination  of  the  proteolytic  activity  at  a  concentration  of 
0.5  per  cent.  Na,C03,  3  c.c.  of  a  sodium  carbonate  solution  (containing 
0.125  gramme  Na2COs)  was  used  instead  of  the  N/2  hydrochloric  acid. 
For  the  determination  of  the  proteolytic  activity  at  the  alkalinity  of  the'  egg 
3  c.c.  of  salt  solution  was  used. 
