ENZYMES. FERMENTATION 419 



. II. Invertase, Emulsin. 



In measuring the activity of these enzymes either the reduction is estimated, 

 preferably after removing proteins as under lactase, or the change in optical 

 activity is observed. 



A known weight of preparation is dissolved or extracted with water and 

 made up to a definite volume. 



i to 5 c.c. or other suitable volume of the solution or extract, is added 

 to 100 c.c. of 2-5 per cent, substrate solution. The solution is observed 

 in the polarimeter at once and at definite intervals and the readings noted ; 

 or after a fixed interval of time at room temperature, or at 37, the action is 

 stopped with 10 c.c. of -iN alkali, the volume made up to 200 or 250 c.c., 

 and the reduction measured (a) against 5 or 10 c.c. of Fehling's solution, or 

 (b) gravimetrically. 



III. Lactase, Maltase. 



The measurement of the activity of lactase and maltase on account of 

 the small differences in reduction, and rotation also in the case of lactase, is 

 somewhat tedious. A control experiment must be performed. Lactase 

 solutions generally contain a large amount of protein if prepared from the in- 

 testine and this must be removed before an estimation can be carried out. 



Two portions of 100 c.c. of 5 per cent, lactose solution are placed in two 

 250 c.c. flasks. To each 50 or 100 c.c. of lactase solution or intestinal ex- 

 tract are added. To one of them (C), the control, 5 or 10 c.c. of neutral 

 mercuric nitrate solution are added. 1 To both are added 5 c.c. of toluene. 

 They are kept in corked flasks at 37 for 3-4 days. (Instead of adding 

 mercuric nitrate to (C), 50 c.c. of boiled and cooled enzyme solution might 

 have been added and mercuric nitrate added to both after 3-4 days.) The 

 same volume of mercuric nitrate solution is added to the other flask contain- 

 ing enzyme (E) and mixed. The two flasks are now in the same stage of 

 operations and their contents are treated as follows : 



(1) Filtered through dry papers into dry flasks. 



(2) Equal volumes of filtrate (as much as possible) are taken and 

 neutralised to litmus with sodium hydroxide from a burette (about 4-8 c.c.). 

 The same quantity is added to both. 



(3) The precipitate is filtered off through a dry paper and the filtrate 

 collected in a dry flask. 



(4) Equal volumes of filtrate (as much as possible) are taken and treated 

 with hydrogen sulphide. .The gas is passed on to the surface of the solution. 

 Only a small quantity is required to remove the last traces of mercury. 



(5) Excess of hydrogen sulphide is removed by adding copper sulphate 

 solution until the smell disappears and it has a faint blue colour. 



(6) The solution is filtered from the sulphides and the volume is made up 

 to 250 or 500 c.c. 



The reducing sugar is estimated gravimetrically or by Bertrand's method. 



The percentage of hydrolysis is calculated as follows : 



The reducing power of the control multiplied by - is the reducing 

 power of lactose if completely hydrolysed. The difference between this 

 figure and the original is that of total hydrolysis T. 



The difference between the control figure and that of the enzyme solution 

 is the actual hydrolysis A. 



Hence percentage is A : T = x : 100. 



1 220 gm. of mercuric oxide are suspended in about 200 c.c. of water and dissolved by 

 adding concentrated nitric acid. The solution is treated with caustic soda until a per- 

 manent precipitate of mercuric oxide is formed. The solution is filtered and made up to 



id * 



1000 C.C. 



