io6 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



crystallises out. 200 c.c. of alcohol are added, and after standing for 

 one hour the salt is filtered off and washed with 80 per cent, alcohol. 

 The salt is dissolved in water and the nitrogen estimated ; usually 

 very little nitrogen is contained in it, but if it be large in amount, the 

 solution is acidified, evaporated, and treated with alcohol as described. 



The alcoholic solution is evaporated in vacuo to dryness, dissolved 

 in water and made up to 200 c.c. 



Total nitrogen is determined in 5 c.c., and 25, 30, or 40 c.c., 

 according to the amount of nitrogen, are neutralised with sodium 

 hydroxide, diluted to 50 c.c., and used for the determination of amino 

 nitrogen. 



The mono-aminodicarboxylic acid nitrogen is determined by exactly 

 neutralising 20 or 25 c.c. in a platinum basin to azolitmin paper 1 and 

 evaporated to dryness, the concentrated solution being stirred with a 

 platinum wire if salts crystallise out, and ashed over a spirit burner. 

 The ash is dissolved in not too great an excess of o*2N hydrochloric 

 acid, washed into a conical Jena-glass flask, boiled over a flame for 

 five to ten minutes, cooled and titrated with O'2N alkali with phenol- 

 phthalein as indicator. The difference between the amounts of acid 

 and alkali is equivalent to the carboxyl groups of the mono-amino- 

 dicarboxylic acids and is expressed in terms of nitrogen by multiplying 

 the figure in c.c. by 2 -8. 



14. Corrections for the Solubilities of the Phosphotungstates. 

 Although the conditions of precipitation are not always the same, the 

 variation is not sufficient to cause significant change in the solubilities 

 of the phosphotungstates. When precipitated from a volume of 200 

 c.c. the following corrections should be added, and the sum of the 

 figures for amino nitrogen and non-amino nitrogen subtracted from 

 the figures for the mono-amino acids : 



1 Henriques and Sorensen (Zeitschr.physiol. Chem., 64, 133) describe the preparation of 

 sensitive litmus paper as follows : 



0-5 gram of powdered azolitmin is dissolved in 200 c.c. water + 22*5 c.c. 'iN NaOH 

 in a basin. 50 c.c. of alcohol are added after filtering. Strips of good, ash-less filter paper 

 are drawn through the solution and dried by hanging on strings. The preparation takes 

 about one hour. The paper must behave in the following way with Sorensen's phos- 

 phate solutions : 



To 3 sec + 7 prim (P H = 6, 47) slightly acid reaction. 



To 5 sec + 5 prim (Pn = 6, 81) neutral reaction. 



To 7 sec + 3 prim (Pn* = 7-17) slightly alkaline reaction. 



The neutral point is chosen slightly more on the acid side (= 7-07) on account of 

 ammonium salts and amino acids. 



If a litmus paper does not react in this way, the amount of iN NaOH used in its 

 preparation must be altered accordingly. 



