ENZYMES. FERMENTATION 425 



(d) Sorensen's Method. 



The most accurate and simple method is that of Sorensen. In 

 this method the rate of formation of the products of the action of the 

 enzyme, namely, the amino acids, is measured. The amino acids 

 contain both a carboxyl group and an amino group and consequently 

 their reaction is neutral. By combining the amino group with formalde- 

 hyde, its basic character is destroyed and the carboxyl group is free 

 to exert its acid character. The reaction which takes place is : 



R . CH . NH 2 + OHCH R . CH . N : CH 2 + H 2 O 

 COOH COOH. 



Samples of a trypsin digest of caseinogen, gelatin, etc., are treated 

 at intervals with neutral formaldehyde. They show a gradual in- 

 crease in acidity as the action of the enzyme proceeds ; the rate of the 

 increase depends on the strength of the enzyme. 



60 c.c. of formalin are diluted with two volumes of water and 

 neutralised by running in *iN alkali from a burette until the colour is 

 just red to phenolphthalein, which is added as indicator. 



100 c.c. of a 4 per cent, caseinogen solution in 0*4 per cent, 

 sodium carbonate solution are measured out into a smail flask, warmed 

 to 40 and then 5 c.c. or more of the trypsin solution are added ; the 

 mixture is kept at 40. Immediately after the addition a sample of 

 25 c.c. is removed with a pipette and 30 c.c. of the previously 

 neutralised formaldehyde solution are added. At intervals of half an 

 hour, one hour, one hour and a half, two hours, further samples of 25 

 c.c. are removed and to them are added 30 c.c. of the formaldehyde 

 solution and a few drops of phenolphthalein. Each sample as it is 

 obtained is titrated with the -iN alkali in the burette until the solution 

 has a distinctly red colour. The amount of alkali used for each 

 sample is noted. 



(e) Method by Estimating the Nitrogen of the Amino Acids. 

 Proteins are precipitated by the various alkaloidal reagents. These 



reagents are of use in measuring the action of trypsin. Most frequently 

 tannic acid is used, but trichloracetic acid and phosphotungstic acid are also 

 employed. 



100 c.c. of a solution of protein, as in Sorensen's method, are mixed 

 with a known amount of trypsin solution and digested for 2-48 hours in the 

 presence of toluene. Immediately after mixing and at intervals of 15, 30, 

 or 60 minutes a sample of 10 c.c. is removed and put into 10 c.c. of tannic 

 acid or trichloracetic acid solution. When the precipitate has settled the 

 solution is filtered through a dry paper into a dry vessel. A nitrogen 

 estimation by Kjeldahl's method is made with 5 C.C. or any aliquot portion 

 of the filtrate. 



(/) Physical Methods. 



Several physical methods can be used to estimate trypsin, but they are 

 adapted mainly for the study of the catalytic action. The change in electri- 

 cal conductivity, in viscosity, in rotation has been frequently observed ic 

 experiments with trypsin. 



