Ill] PLANT ENZYMES - 25 



Expt. 15. Action of catalase. (Harden and Zilva, 12.) Completely fill a test-tube 

 with hydrogen peroxide (20 vols.) solution which has been diluted with an equal 

 volume of water and add 0*5-1 gm. of zymin. Place the thumb firmly over the mouth 

 of the tube, invert and place the mouth under water in a small basin, clamping the 

 tube in position. A rapid evolution of oxygen takes place. When the tube is about 

 three-fourths full of gas, close the mouth with the thumb while still under water and 

 remove the tube. Plunge a glowing splint into the gas and it will re-kindle to a flame. 



Expt. 16. Action of protease. Weigh out 10 gms. of white flour, and allow it to 

 extract with 100 c.c. of distilled water for one hour, shaking from time to time. Then 

 filter on a filter-pump. The extract will contain the albumin, leucosin (see p. 138). 

 Into small flasks {a) and (6) put the following : 



(a) 40 c.c. of the flour extract + 1 gm. of zymin -f 1 c.c. of toluol. 



{h) 40 c.c. of flour extract -\- 1 gm. of boiled zymin -j- 1 c.c, of toluol. 



Shake both flasks, plug with cotton-wool and place them in an incubator at 38° C. 

 for 48 hrs. After incubation, boil the liquid in both flasks, in order to coagulate un- 

 altered protein, and filter. Cool the filtrates from the respective flasks and add 

 bromine water drop by drop (see p. 153). A pink, or purplish-pink colour, due to the 

 presence of tryptophane, will be formed in tube (a). Hence hydrolysis of protein has 

 taken place. Tube (6) will show no colour or only that due to bromine. Add a little 

 amyl alcohol to both tubes and shake gently. The alcohol will be coloured pink or 

 purplish in the tube giving the tryptophane reaction. 



Expt. 17. Action of reductase. (Harden and Norris, 11.) Take two test-tubes, 

 {a) and (6), provided with well-fitting corks and put in the following : 



{a) 1 gm. of zymin -1- 20 c.c. of distilled water -\- 0-5 c.c. of methylene blue solu- 

 tion (made by diluting 5 c.c. of a saturated alcoholic solution to 200 c.c. with distilled 

 water). 



(6) 1 gm. of boiled zymin -|- 20 c.c. of distilled water 4- 0*5 c.c. of methylene blue 

 solution. 



Cork both tubes after adding a few drops of toluol and place in an incubator at 

 38° C. for 1-3 hours. The blue colour will practically disappear from tube {a) but 

 will remain in tube (6). 



The methylene blue is reduced to a colourless leuco-compound which will become 

 blue again on re-oxidation. 



Expt. 18. Enzyme actions of an aqueous extract of zymin. Weigh out 2 gms. of 

 zymin and place them. on a double folded filter-paper in a funnel and wash with 80 c.c. 

 of distilled water. With the filtrate make the following experiments. 



(A) Action of invertase. (Harden and Zilva, 12.) Into two small flasks (a) and (b) 

 put the following : 



{a) 10 c.c. of a 2 % solution of pure cane-sugar -f- 10 c.c. of the filtrate from zymin. 



(6) 10 c.c. of the same solution of cane-sugar -h 10 c.c. of the boiled filtrate from 

 zymin. 



Put both flasks in an incubator at 38° C. After 30 minutes add equal quantities 

 (about 1-2 c.c.) of Fehling's solution to both flasks and boil (see p. 54). Flask {a) 

 will show considerable reduction of the Fehling. Flask {h) will show comparatively 

 little reduction, that which does take place probably being due to the sugar previously 

 formed by the action of glycogenase on stored glycogen. 



