PYROCATECHOL GROUPING 97 



its insolubility in alcohol is precipitated on the potato residue. 

 This potato residue no longer turns blue guaiacum directly, 

 but does so on the addition of hydrogen peroxide. In order 

 to demonstrate the presence of the pyrocatechol grouping in 

 the alcohol soluble extract, the following procedure may be 

 adopted. Boil 500 grams of slices of freshly-peeled potato 

 for a quarter of an hour in 250 c.c. of 96 per cent alcohol 

 over a water bath. After filtering, evaporate off the alcohol 

 and extract the residue with a little warm water and filter. 

 To the filtrate add a concentrated solution of lead acetate 

 until no further precipitate is formed. Filter off the precipitate 

 and after washing, suspend it in a little water and add 10 

 per cent sulphuric acid drop by drop until the yellow colour 

 of the precipitate turns white owing to the formation of lead 

 sulphate. Filter and carefully neutralize the filtrate with I 

 per cent caustic soda. The presence of the pyrocatechol 

 grouping may be demonstrated by the addition of a drop 

 of ferric chloride which produces a green colour; on adding 

 a few drops of I per cent sodium carbonate, the colour changes 

 to blue and then to reddish purple. According to Wheldale 

 Onslow it is the presence of this pyrocatechol group in the 

 substrate which provides the peroxide for the peroxidase to 

 activate and there is consequently no need to postulate the 

 existence of a second enzyme or oxygenase as is assumed by 

 Bach and Chodat. The existence of an oxygenase also is 

 disputed by Moore and Whitley.* 



In addition to guaiacum, other reagents have been used 

 in the investigation of oxidizing enzymes ; amongst these may 

 be mentioned benzidene, a-naphthol, /-phenylene diamine, 

 and pyrogallol ; none of these, however, produce any colour 

 with plant tissues or extracts unless hydrogen peroxide be 

 added. This is a very significant fact, inasmuch as all oxidiz- 

 ing enzymes found in plants would appear to be peroxidases 

 as tested by these reagents and the distinction between oxidase 

 and peroxidase would therefore appear to be dependent upon 

 the reagent employed. Contrariwise the discovery is within 

 the bounds of possibility of a reagent of sufficient delicacy 

 to be changed in colour by direct action not only by plant 



* Moore and Whitley : " Biochem. Journ.," 1909, 4, 136. 

 VOL. II. 7 



