Ill] ENZYME ACTION 23 



of benzidine solution + 2-3 drops of liydrogen peroxide. (The zyniin is washed by 

 I)utting it on a double folded tilter-i)aper in a funnel and adding distilled water from 

 time to time. 50 c.c. of water should be used for 0-5 gm. of zymin.) 



(d) A susjjension of 0"5 gm. of washed zymin in 10 c.c. of washings + 1 c.c. of 

 benzidine solution + 2-3 drops of hydrogen peroxide. 



A blue colour will develop in (a) showing thtit fresh yeast contains a peroxidase 

 (see p. 109). A l)lue colour will also develop in (c) but not in (6) and (d). This is 

 explained by assuming that the zymin contains an inhibitor, not present in fresh 

 yeast, but which is developed during the preparation of the zymin, and that this 

 inhibitor can be washed away by water. On adding the washings to the washed 

 zymin the reaction is inhibited again. 



Kvpt. 14. 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, clamp- 

 ing 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 thiuiab while still under 

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

 to a flame. 



Expt. 15. 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. 124). Into small flasks (a) and {h) put the following: 



{a) 40 c.c. of the flour extract -I- 1 gm. of zymin + 1 c.c. of toluol. 



(6) 40 c.c. of flour extract -f 1 gm. of boiled zymin + 1 c.c. of toluol. 



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

 for 48 hrs. After incubation, boil the liquid in both tubes, in order to coagulate 

 unaltered protein, and filter. To the filtrates of the respective tubes add bromine 

 water drop by drop (see p. 138). A pink, or purplish-pink colour, due to the presence 

 of tryptophane, will be formed in tube {(t). 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. The alcohol will be coloured pink or purplish in 

 the tube giving the tryptophane reaction. 



Expt. 16. Action of reductase. (Harden and Norris, 11.) Take two small flask.s, 

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



(a) 1 gm. of zymin + 20 c.c. of distilled water + 0-5 c.c. of methylene blue 

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

 distilled water). 



{h) 1 gm. of boiled zymin -}- 20 c.c. of distilled water -f O'o c.c. of methylene blue 

 solution. 



Cork both tuV)es after adding a few drops of toluol and place in an incubator at 

 38° C. for 3 hours. The blue colour will practically disappear from tube («) but will 

 remain in tube (6). 



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

 blue again on re-oxidation. 



