110 AROMATIC COMPOUNDS AND [CH. 



The oxidation of pyrogallol, in the presence of a peroxidase and 

 hydrogen peroxide, has been used as a method for estimating the activity 

 of these enzymes. Solutions of known strength of pyrogallol and hydro- 

 gen peroxide are used, and to the mixture a solution of a known weight 

 of prepared peroxidase is added. An oxidation product, termed purpuro- 

 gallin, is formed. After a definite time, the reaction is stopped by 

 adding acid, and the purpurogallin extracted by ether. The ether 

 extract is colorimetrically compared with an extract containing a known 

 amount of purpurogallin (Willstatter and Stoll, 47). 



Expt. 117. Outline of method for estimating peroxidase by formation of purpuro- 

 gallin. Make a solution of 0'5 gm. of pyrogallol in 200 c.c. of distilled water, and 

 add to it 1 c.c. of 5 % hydrogen peroxide. Then add about 5 c.c. of a solution of 

 Horse-radish peroxidase from Expt. 116. After 5 minutes add to half the mixture 

 25 c.c. of dilute sulphuric acid and extract the purpurogallin with ether in a 

 separating funnel. The purpurogallin will be extracted by the ether, giving a yellow 

 solution. Allow the other half of the mixture to stand. The colour will deepen, and 

 a reddish deposit of purpurogallin will be precipitated. Examine a little of the 

 deposit under the microscope. It will be found to consist of sheaves of crystals. 



A solution of peroxidase from Atyssum leaves [Expt. 119 (&)] can also be used. 



It has recently been shown (Wheldale Onslow, 11) that the per- 

 oxidase element (Expt. 118) of certain oxidases (fruit of Pear, tuber of 

 Potato) can directly oxidize the phenol catechol (see p. 88), as well 

 as other substances containing the catechol grouping, that is two 

 hydroxyl groups in the ortho position, e.g. protocatechuic acid and caffeic 



acid: 



COOH CH=CH'COOH 



OH 



OH OH OH 



Catechol Protocatechuic acid Caffeic acid 



A brown colour is produced, giving rise to some oxidation product 

 which will then act as a peroxide, the peroxidase being present, to form 

 the system (oxidase) which will blue guaiacum. 



It appears probable that the above phenomenon, as illustrated in 

 the Pear and Potato, affords an explanation of the reaction of the oxidiz- 

 ing enzymes of all plants which give the oxidase reaction and turn brown 

 on injury. For such plants usually contain an aromatic substance 

 with the catechol grouping. On the death of the cell (which may be 

 brought about, either by injury, or treatment with chloroform vapour, 

 i.e. on autolysis, see p. 19) this aromatic substance is oxidized by the 



