20 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(2) Color imetric Estimation. 



Folin and Denis [1912, 2] introduced a method for the quantita- 

 tive estimation of tyrosine in proteins depending upon the blue colour 

 which tyrosine gives with a phosphotungstic-phosphomolybdic reagent 

 [1912, i]. 



The estimation is carried out as follows : 



A weighed quantity about I gram of dried protein ishydrolysed 

 by boiling under a reflux condenser for twelve hours with 25 c.c. of 

 20 per cent, hydrochloric acid. The solution is transferred to a 100 

 c.c. measuring flask and made up to volume. One or two c.c. of this 

 solution are placed in a 100 c.c. flask, 5 c.c. of the reagent x are added, 

 and after five minutes 25 c.c. of a saturated solution of sodium car- 

 bonate. The volume is made up to 100 c.c. with water. At the same 

 time 5 c.c. (= I mgm.) of a standard tyrosine solution 2 are placed 

 in another 100 c.c. measuring flask and treated in the same way. The 

 maximum blue colour develops in half an hour. After this time the 

 colours of the two solutions are matched in a Duboscq colorimeter, 

 the standard being set at 20 mm. The solutions, if not clear, are 

 filtered before making the comparisons. 



Folin and Denis' results, compared with the gravimetric data, 

 were : 



Wool (sheep) . 

 Hair (human 

 Horn (cow) 

 Gelatin 

 Ovomucoid 



Globulin (flax seed) . 

 Glycinin (soy bean) . 

 Phaseolin (white bean) 

 Globulin (squash seed) 

 Corylin (hazel nut) 

 Edestin (hemp seed) . 

 Amandin (almond) 

 Globulin (cotton seed) 



Caseinogen 

 Vitellin 

 Conalbumin 

 Ovalbumin 

 Lactalbumin 

 Globulin (castor bean 

 Vignin (cow pea) 

 Legumin (pea) 

 Glutelin (maize) 

 Zein (maize) 

 Hordein (barley) 

 Gliadin (wheat) 

 Glutenin (wheat) 



In all cases the values by the colorimetric method are higher than 

 those by the gravimetric. These higher values for tyrosine were shown 

 by Folin and Denis not to be due to the reaction taking place with 



1 ioo grams of sodium tungstate, 20 grams of phosphomolybdic acid and 50 c.c. of 85 

 per cent, phosphoric acid are added to 750 c.c. of water. The solution is boiled for two 

 hours under a reflux condenser, cooled and diluted to 1000 c.c. 2 c.c. of this reagent will 

 give the maximum colour with i mgm. of tyrosine. 



2 Pure tyrosine dissolved in -iN HC1 so that 5 c.c. contain i mgm. of tyrosine. 



