24 PLANT ENZYMES [ch. 



Expt. 12. Action of maltase. (Harden and Zilva, 12.) Into each of two small 

 flasks, put 20 c.c. of a 2 7o solution of maltose and 0*5 gm. of zymin. Boil the 

 contents of one flask. Then plug both flasks with cotton -wool, add a few drops of 

 toluol and place in an incubator at 38° C. for 12-24 hours. Filter the liquid from 

 both flasks and test by making the osazone (see p. 50), using at least 10 c.c. of the 

 filtrate in each case. Glucosazone will crystallize out from the unboiled, maltosazone 

 from the boiled, mixture. 



Expt. 13. Action of carboxylase. (Harden, 10.) The action of carboxylase on 

 pyruvic acid is detected by the formation of carbon dioxide and acetaldehyde. Care- 

 fully prepared zymin will still respire, but, after washing, some constituent essential 

 to respiration is removed. Hence the zymin must be first washed and tested. Take 

 5 gms. of zymin and wash well on a filter with distilled water. Then suspend the 

 zymin in 50 c.c. of water in a flask and draw a slow current of air (previously passed 

 through two bottles of strong caustic soda and two bottles of saturated baryta 

 solution) through the suspension of zyniin into a receiving flask of baryta solution. 

 The flasks should be connected with pressure tubing and the apparatus must be air 

 tight. Continue to draw the current of air through until it ceases to produce a milki 

 ness in the receiving flask, due to any carbon dioxide in solution or to residual 

 respiration. Then add quickly to the suspension of zymin 50 c.c. of 1 % pyruvic acid 

 (by weight), 5 c.c. of normal caustic potash and 6 gms. of boric acid ; also a few drops 

 of caprylic alcohol to prevent frothing. Place the flask in a beaker of water at 

 30-40° C. and again draw a current of air. A copious precipitate of barium carbonate 

 will be formed in the receiving flask. The boric acid is used to prevent the solution 

 from becoming too alkaline owing to the formation of potassium carbonate, and, 

 being a weak acid, it has no inhibiting action on the enzyme. 



The contents of the flask containing the zymin are filtered into a small distilling 

 flask and about 10 c.c. of distillate collected (cooled with ice if possible). To this 

 add 1-2 c.c. of a freshly made 1 ^Jq solution of sodium nitroprusside, followed by a 

 few drops of piperidine. A deep blue colour denotes the presence of acetaldehyde. 



Expt. 14. Action of peroxidase (Harden and Zilva, 12.) Into four small 

 evaporating dishes, (a), (6), (c) and (c^), put the following : 



(a) A suspension of 0*5 gm. of fresh yeast in 10 c.c. distilled water -I- 1 c.c. of 

 benzidine solution (1 o^ in 50% alcohol) + 2-3 drops of hydrogen peroxide (20 vols.). 



(5) A suspension of 0*5 gm. of zymin in 10 c.c. of distilled water + 1 c.c. of 

 benzidine solution + 2-3 drops of hydrogen peroxide. 



(c) A suspension of 0*5 gm. of washed zymin in 10 c.c. of distilled water+1 c.c. 

 of benzidine solution + 2-3 drops of hydrogen peroxide. (The zymin is washed by 

 putting it on a double folded filter-paper 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 suspension 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 that fresh yeast contains a peroxidase 

 (see p. 124). A blue colour will also develop in (c) but not in {h) 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. 



