126 . AKOMATIC COMPOUNDS [ch. 



residue pressed free from alcohol in a press. The residue is next extracted with 40 % 

 alcohol for 48 hrs., filtered and precipitated with 90 % alcohol. The precipitate, 

 which contains the peroxidase, is filtered off". Dissolve up in water and make the 

 test for peroxidases (Expt. 118). 



Peroxidase from the Horse-radish has been prepared on a large scale 

 and very carefully purified (Willstatter and Stoll, 34). The purified 



t product was found to consist chiefly of a nitrogenous glucoside, a result 

 which does not throw much light on its catalyzing properties. 



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, 34). 



Expt. 122. Outline of method for estimating peroxidase hy 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 o/o hydrogen peroxide. Then add about 5 c.c. of a solution of 

 Horse-radish peroxidase from Expt. 121. 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 Alyssum leaves [Expt. 124 (6)] can also be used. 



The fact that an oxidase contains an oxygenase and catechol substance 

 may be demonstrated as follows. The tissue of an oxidase plant is rapidly 

 pounded under alcohol (to avoid oxidation) and extracted several times 

 with cold alcohol, by which the. catechol substance is removed. The two 

 enzymes, oxygenase and peroxidase, remain in the tissue residue. This 

 residue or its water extract will give no (or very little) reaction with 

 guaiacum, since one of the components for producing the peroxide has 

 been removed. If now a little catechol is added followed by guaiacum, 

 a blue colour immediately appears. Moreover, from an alcoholic extract 

 of the tissues the catechol substance can be precipitated as a lead salt, 

 the lead removed as insoluble sulphate, and the aromatic compound set 

 free again in solution. If the enzyme extract is then added to the solu- 

 tion of the catechol substance, a brown colour is produced together with 

 a peroxide, and the mixture will give a blue colour with guaiacum. 



