424 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



B. Trypsin. 



(a) Metis Method. 



The activity of a trypsin solution can be measured by Mett's method in 

 the same way as described for pepsin. The tubes may contain either egg- 

 white or gelatin (p. 409). 5 c.c. of "4 per cent, sodium carbonate solution 

 and 5 c.c. of the trypsin are placed in a small conical flask together with i or 

 2 Mett's tubes, i c.c. of toluene, or chloroform, is also put in the flask as 

 antiseptic. After periods of 2, 4, 8-24 hours the lengths digested from each 9 

 end are measured. 



The squares of the lengths digested more nearly represent the activity 

 than the actual lengths which are measured. 



(b) Roberts' Method. 



The action of trypsin upon milk serves as a very convenient and 

 simple property for determining the activity of the enzyme. Roberts 

 in 1 88 1 discovered that trypsin acting upon milk produced a striking 

 change in its properties at an early stage of its digestion, namely, 

 coagulation on heating due to the formation of casein (metacasein as 

 it was termed). This property disappears at the end of the digestion. 



30 c.c. of milk are diluted with 30 c.c. of water in a small flask 

 and warmed to 40; 1-5 c.c. of the trypsin solution are added. The 

 time is noticed and at the end of every minute a portion of 5 c.c. is 

 withdrawn and heated to boiling. It is noticed at what time a 

 distinct curdling or precipitation occurs. A sample on heating shows 

 incipient curdling before the actual appearance of the curdling ; this 

 indicates that the next sample will probably coagulate properly. 



The activities of trypsin solutions are proportional to the time 

 taken to produce curdling. 



Vernori l considers this method very exact if the values are cor- 

 rected by reference to a table. 



(<:) Fuld- Gross Method. 



Another convenient method of measuring the activity of trypsin solutions is 

 that of Fuld-Gross. A caseinogen solution is digested with trypsin ; acidulated 

 alcohol is added to precipitate unchanged caseinogen after a certain time. 



The substrate is prepared by dissolving -i gm. of pure caseinogen in 5 c.c. 

 of 'iN sodium hydroxide + 25 c.c. of water and heating to boiling. The 

 solution is cooled ; about 4-5 c.c. of -iN HCl are added to neutralise the excess 

 of alkali and the volume is made up to 100 c.c. It keeps for 48 hours ; 

 it is not advisable to add toluene which tends to precipitate the caseinogen. 



The acid solution is prepared by mixing together i part of glacial acetic 

 acid, 49 parts of water and 50 parts of 96 per cent, alcohol. 



Ten test tubes are filled in series with increasing amounts of trypsin solu- 

 tion -oi to i c.c. Water is added to make the volumes, where necessary, 

 equal to i c.c. 2 c.c. of the caseinogen solution are added to each tube and 

 they are placed for i hour in a bath at 37. They are removed and 6 drops 

 of the acid alcohol added to each. In those tubes containing undigested 

 caseinogen there is a flocculent precipitate. The first tube containing no 

 precipitate in which there is the smallest amount of enzyme is noticed. The 

 result is calculated according to the number of c.c. of caseinogen solution 

 which can be digested by i c.c. of enzyme solution : thus supposing it were '2. 

 02 : 2 = i : x x = 100 



The trypsin solution is spoken of as containing 10 units. 



. * J. Physiol., 27, 182. 





