viii] AROMATIC COMPOUNDS 127 



Expt. 123. Resolution of the components of the oxidase in the Potato tuber. (A) 

 Separation of peroxidase and oxygenase. Cut a few thin slices from a peeled potato 

 and put them in a mortar which contains sufficient 96 % alcohol to prevent, as far 

 as possible, exposure to the air, and pound them thoroughly. Filter quickly on a 

 filter-i)ump, and repeat the process several times until a colourless powder, consisting 

 of cell-residues, starch, etc. is obtained. The enzymes (including the peroxidase and 

 oxygenase) of the cells are precipitated by the alcohol and remain in the cell-residue. 

 Make a water extract of the white powder and filter. To a portion of the filtrate add 

 a few drops of guaiacum tincture ; no blue colour is given. Add further a few drops 

 of dilute hydrogen peroxide: a blue colour appears. (B) Separation of the aromatic 

 substance. Take about 500 gms. of freshly peeled potato tuber, cut it into thin slices 

 and drop them as rapidly as possible into a flask containing 250 c.c. of boiling 96 <)/o 

 alcohol on a water-bath. Continue boiling for 15 mins,, and then filter. Evaporate 

 off the alcohol from the filtrate, take up the residue in a little water, warm and filter. 

 To the filtrate add concentrated lead acetate solution until a precipitaoe ceases to be 

 formed. Filter off the precipitate, which is pale yellow in colour, stir up in a little 

 water and add 10 % sulphuric acid drop by drop until the yellow colour is destroyed, 

 and the lead is converted into lead sulphate. Filter off the lead sulphate : the filtrate 

 contains the aromatic substance in solution. Neutralize the solution carefully with 

 1 ^/o caustic soda and make the following tests with separate portions in small 

 evaporating dishes : 



(a) Add a drop of ferric chloride solution : a deep green colour appears. Add 

 further a few drops of 1 o/^ sodium carbonate solution. The green colour changes to 

 a bluish- and finally, a reddish-purple. This reaction is characteristic of aromatic 

 compounds containing the catechol grouping, i.e. two hydroxyl groups in. the ortho 

 position (see p. 123). 



(6) Add a little of the enzyme solution prepared in (A). The mixture will 

 gradually turn brown owing to the oxidation of the aromatic by the oxygenase. 



(c) To (6) add a few drops of guaiacum tincture. A blue colour is given owing to 

 the presence of the peroxide formed in (6), the oxidase system being now complete. 



Expt. 124. Actio7i of oxygenase on catechol, (a) The oxygenase of the Potato 

 tuber {or Pear fruit). Make a 1 o/q solution of catechol in distilled water. To some 

 of this solution, in a small evaporating dish, add a little of the enzyme solution from 

 Expt. 123 (A). Note that the catechol solution gradually turns brown. Add further 

 a few drops of guaiacum tincture. A blue colour appears, (b) Enzyme extract of 

 Alyssum leaves. Pound up 2-3 Alyssum leaves in a mortar with some 96 % alcohol, 

 and filter on a filter-pump. Repeat the process until the residue is practically 

 colourless. Extract the residue with a little distilled water and filter. Proceed as in 

 (a). No browning of catechol takes place and no blue colour is formed on the sub- 

 sequent addition of guaiacum. 



For section (a) the following material may also be used: fruits of Apple and 

 Greengage, flowers of Horse Chestnut (Aesculus) and leaves of Pear, the method of 

 preparation in (6) being employed. For section (6) flowers of white Arabis may also 

 be used. 



If in the preparation of the enzymes from the Potato tuber, the 

 tissue is allowed to brow^n before extracting with alcohol, the cell-residue 



