214 COMPOSITION OF THE DIGESTIVE JUICES. [CH. VIII. 



as calcium paracaseinate if there be a sufficient concentration of calcium salts 

 present, as there is in Ex. 257. The later products of hydrolysis are bodies 

 allied to the proteoses (caseoses), peptones, and finally the ammo-acids. The 

 failure to obtain a precipitate with acetic acid marks the stage when the last 

 trace of paracasein has been hydrolysed. Though rennin and pepsin in 

 neutral or faintly acid solution can hydrolyse casein to paracasein, they are 

 unable to effect the further stages of break down, and therefore do not give 

 this important test for casein. 



259. Estimation of Trypsin. Having demonstrated that a 

 given solution contains trypsin, this can be estimated by following 

 the procedure given in Ex. 252, as first suggested by J. Mellanby. 

 The same unit as that adopted for pepsin is convenient. 



260. The course of tryptic digestion of casein as followed by 

 formol titrations. 



Principle of the method. Suppose the constitution of a protein to be 

 represented by the following formula : 



R R x R 2 R 3 



I I I 



.CH. 



H 2 N.CH.CO - NH.CH.CO - NH.CH.CO - NH.CH.COOH. 



The aqueous solution of such a compound would probably be nearly neutral, 

 the acidity due to the terminal - COOH group being balanced by the basicity 

 due to the terminal - NH 2 . The addition of a small amount of soda would 

 render the solution alkaline to phenol phthalein. On adding a neutralised 

 solution of commercial formaldehyde (formol) the basicity of the - NH 2 

 group is removed by the formation of a methylene group [see p. 69 (3)]. 



- NH 2 + OHC.H = - N : CH 2 + H.,0. 



The whole molecule would now behave as an acid owing to the unopposed 

 influence of the terminal - COOH group ; and the solution would require the 

 addition of an equivalent of soda to make it again alkaline to phenol phthalein. 

 Let this amount of soda be A. 



If the protein be hydrolysed by an enzyme into its constituent amino- 

 acids, the amount of soda required for neutralisation would probably be 

 approximately the same as that for the intact protein. But after treatment 

 with formol the amount required to make the solution alkaline to phenol 

 phthalein would be four times greater than A. The reason for this is that after 

 hydrolysis there are four free amino-groups and four free carboxylic groups, 

 and the solution is still about neutral. But after treatment with formol the 

 basic influence of all four amino groups is removed, and to make the solution 

 alkaline enough soda has to be added to neutralise all four carboxylic groups. 

 It therefore follows that the degree of hydrolysis of such a protein can be 

 followed by determining the amount of standard alkali required to neutralise 

 the solution after treatment with formol. 



The usual method adopted is to withdraw samples of the digesting 

 mixture at intervals, add neutralised formol and titrate to a definite pink colour 

 to phenol phthalein. The amount required (less the amount for a sample 

 taken immediately after the addition of the enzyme) is a measure of the number 

 of amino-groups set free. In some cases the solution is neutralised to litmus 

 before the addition of the formol. A practical difficulty is that of defining the 

 exact end point of the titration so that the same final reaction is obtained in 



