ENZYMES. FERMENTATION 421 



Proteoclastic Enzymes. 



The numerous methods which have been employed for comparing 

 the strengths of two proteoclastic enzymes may be divided into two 

 main groups. 



I. Those in which the action of the enzyme is determined by ob- 

 serving the rate of solution of an insoluble substrate. 



II. Those in which the rate of formation of the products of the 

 action of the enzyme is observed. 



Of these the former is generally used for pepsin, the latter for trypsin. 

 A. Pepsin. 



(a) Bruckes Method. 



In this method the time taken to dissolve'' equ&l-sized threads of fibrin 

 is noted. The more active enzyme produces solution in the shorter time. 



(b) Metfs Method. 



Numerous results, especially those in Pavloff s laboratory, upon digestion 

 by enzymes have been obtained by this method. It consists in directly 

 measuring the amount of protein digested in a given time, the protein being 

 contained in narrow tubes open at both ends and known as Mett's tubes. 



Mett's tubes consist of a small length of coagulated egg-white or serum 

 in a narrow glass tube of 2 mm. bore, and are made by drawing up egg-white 

 into the glass tubing (no air bubbles must be present in the egg-white) and 

 placing it in nearly boiling water for 2-3 minutes. This tubing is then 

 cut up into lengths of about i cm. The coagulated egg-albumin! tnust form 

 a continuous layer free from air spaces. Two small pieces of tube are placed 

 in the enzyme solution and after a definite lapse of time the pieces are laid 

 upon a mm. scale and the amount dissolved from each end measured. The 

 mean of these readings is taken. 



5 c.c. of 0-4 per cent, hydrochloric acid, 5 c.c. of pepsin solution and 

 i or 2 Mett's tubes are placed in a small conical flask. The flask is stoppered 

 and kept at 37 for 8-10 hours. It is then removed and the amount of pro- 

 tein digested from each end of the Mett's tubes is measured. The mean is 

 taken. In this experiment where the action is for a long period of time, ac- 

 cording to the Schiitz law the squares of the lengths digested represent the 

 activity of the enzyme more accurately than the direct ratio. 



(c) Griitzner's Method, 



This method is the one which has been most frequently em- 

 ployed on account of the rapidity with which the results are obtained. 

 Fibrin, stained with carmine, is the substrate used ; a definite quantity 

 is added to the enzyme solution and according to the rate of diges- 

 tion of the fibrin more or less of the dye-stuff passes into solution. 

 Comparison is made by observing the depth of the colour. 



Roaf has suggested the use of fibrin stained with congo red instead 

 of carmine. This possesses the advantage that the stained fibrin can 

 be used in both acid and alkaline media ; carmine-stained fibrin can 

 only be used in acid media as carmine is dissolved out of the fibrin by 

 alkalies, 



