62 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



I in 1 20 are made until there is the best matching of the colours. 

 Finally, a stage will be reached at which I in 100 may be the best 

 match and I in 101 will give a colour which is slightly less intense. 

 The final matching may be performed in a Duboscq colorimeter. 



With solutions containing other amino acids, just as with concen- 

 trated solutions of histidine, no colour appears unless more reagent 

 is added. This is due to the combination of other amino acids with 

 the reagent. In order therefore to estimate histidine under these con- 

 ditions more reagent is necessary and in order to avoid excess, which 

 interferes by producing a dirty-green colour, a fixed volume of 20 c.c. 

 was decided upon. This volume allows of an alteration of the amount 

 of diazo reagent of from I -5 to 7 c.c., e.g., 10 c.c. solution + 4*5 c.c. 

 reagent + 3 c.c. soda +2*5 c.c. water. The standard was also diluted 

 to 20 c.c. with 5-5 c.c. water. Two variables are thus introduced in 

 effecting an estimation (a) the necessary dilution of the histidine solu- 

 tion, (&) the necessary amount of reagent. 



The first testing is carried out with the reagent k, a series being 

 prepared represented as k 1 , 6 ' 5 , k? . . . 2 , i 1>5 , where the index denotes 

 the amount of reagent. Testing is then done with dilutions of the 



k b k* 

 solutions, the series being represented by , etc., where the denomi- 



4 5 



nator denotes the dilution. In practice all the preliminary experiments 

 are not necessary, since as soon as the quantity of reagent is found, 

 those trials with amounts greater than this quantity can be omitted. 

 With greater dilution generally less reagent is required. The series 

 experimentally tested may be for example 

 I. k\ k*'\ 5 . . . 3 , 2 - 5 , k\ k 1 '* optimal reaction with k\ 



333333 3' 



TV P' 5 k* k & k & 



~> > > > JJ )) >J 



44444 4 



V. ^, , -, ^. All colours below standard. 



The estimation is then made with the solution represented by ,/'.*., 



4 



with a dilution of I in 4 and with 3 c.c. of reagent. 



