112 AROMATIC COMPOUNDS AND [CH. 



them as rapidly as possible into a flask containing 250 c.c. of boiling 96 % alcohol 

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

 alcohol from the nitrate, take up the residue in a little water, warm and filter. To 

 the filtrate add concentrated lead acetate solution until a precipitate 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 % 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 % 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. 110). 



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

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



(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. 119. Action of peroxidases on catechol. (a) The peroxidase of the Potato 

 tuber (or Pear fruit}. Make a 1/ solution of catechol in distilled water. To some 

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

 from Expt. 118 (A). Note that the catechol solution gradually turns brown. Add 

 further a few drops of guaiacum tincture. A blue colour appears. (6) Peroxidase 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 

 subsequent 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 peroxidase from the Potato tuber, the 

 tissue is allowed to brown before extracting with alcohol, the cell-residue 

 is tinged with brown and, on extraction with water, the nitrate will give 

 an oxidase reaction with guaiacum. This is to be explained by the fact 

 that the peroxide has been adsorbed by the tissue residue. This pheno- 

 menon is probably the explanation of the preparation of some oxidases 

 called " laccases." Such enzymes have been obtained by the precipita- 

 tion with strong alcohol of the expressed juices (containing peroxide 

 since they were obtained by crushing the tissues) of plants which brown 

 on injury. The enzyme and other organic matter is precipitated and 

 carries with it the peroxide. 



