.414 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Demonstration of the Catalytic Action of Enzymes. 



The catalytic action of enzymes is readily followed by analysing the amount 

 of decrease of the substrate or the amount of the products formed at intervals 

 during the progress of the reaction. 



Physical methods of analysis are preferable to chemical methods as they are 

 easier of manipulation, e.g. : 



(1) Optical activity. The rotation of the solution of enzyme and substrate 

 is taken immediately the mixture is made and at intervals of i minute, i hour, 

 i day afterwards. Throughout the experiment a constant temperature must 

 be maintained. 



(2) Electrical conductivity. This method can be employed when electro- 

 lytes are produced as end products, e.g. fatty acids from ethyl butyrate and amino 

 acids by the action of trypsin upon proteins. 



(3) Viscosity. Protein solutions are viscous and the decrease in viscosity 

 can be measured. The results by this method are not so accurate. 



When chemical methods are employed samples of the solution must be re- 

 -moved at the beginning and at intervals from a larger bulk of solution. The 

 action of the enzyme must be stopped immediately after removing the solution. 

 This cannot be effectively accomplished by boiling as it is impossible to 

 raise the temperature of the samples to boiling at the same rate ; it can only 

 be done by pouring the enzyme solution into boiling water. In this case 

 dilution occurs and the manipulation of the solution is troublesome. 



The action of the enzyme is most effectively stopped by adding an excess 

 of alkali or acid. In the case of carbohydrates the mutarotation l of the solu- 

 tion takes place immediately on adding the alkali. Since enzymes are associated 

 with proteins or complex carbohydrates, precipitation with heavy metals or 

 tannic acid is very convenient. A definite volume of the solution is added to 

 a definite volume of the reagent. A known volume of the filtrate is analysed 

 after removing the heavy metal or other reagent. Each sample is analysed 

 in exactly the same way. 



1 The sugar is split off in either the a or & forms ; the equilibrium mixture is thus 

 obtained immediately ; by noticing the change in rotation on adding alkali it can be ascer- 

 tained whether the a or /8 form of the sugar is contained in the substrate. 



