PROTEOLYTIC ENZYMES 



505 



End Point Color Standard. Mix methyl green, fuchsin, and picric 

 acid to obtain the bluish-green color obtained at the titration end 

 point. 



Substrate, (a) Dissolve 12 g. casein in 8 ml. 1 A?" ammonium hy- 

 droxide in the presence of 0.05 ml. octyl alcohol and make up to 

 100 ml. with water, (b) Prepare buffer soln. by mixing 2 N am- 

 monium hydroxide with 2 A'' ammonium chloride in the ratio a/b 

 given in the following table, (c) Prepare a buffer-pH correction 

 soln. for the casein by mixing 1 ml. of the chosen buffer soln. 

 with the corresponding number of milliliters of alkali as in- 

 dicated in Table VII and make up to 10 ml. with water. The so- 

 dium hydroxide may be used instead of the ammonium hydroxide 

 to avoid high concentrations of ammonia, (d) Before the experi- 

 ment mix 1 ml. of the 12% casein soln. with 1 ml. of the buffer- 

 pH correction soln. 



TABLE VII 

 Composition of Ammonia Buffers for Various pH Values 



PROCEDURE 



1-4. These steps are the same as in the preceding method. How- 

 ever, since the absorption of carbon dioxide by the alkaline reaction 

 mixture must be avoided, the tubes are capped with soda lime stop- 

 pers. (Fig. 47, page 171). 



5. Stop the reaction by pipetting in 130 [A. alcohol-thymol blue 

 soln. 



6. Titrate to the bluish-green end point with the 0.05 A^ tetra- 

 methylammonium hydroxide, matching the color to the color stand- 

 ard. Protect from carbon dioxide during the titration by using the 

 paraffin-oiled glass bead arrangement (Fig. 85, page 257), or the 

 soda lime "desiccator" (Fig. 86, page 258). 



