The Formation of the Anthocyan Pigments of Plants. 319 



This pigment is at first limited to the veins, though subsequently the 

 whole petal becomes distinctly coloured. The depth of colour is considerable, 

 and the general aspect of the brown petals resembles closely that produced 

 by the action of benzidine. There seems no doubt, indeed, that the brown 

 coloration obtained by treating petals of Lychnis coronaria with an alcoholic 

 solution of benzidine is due to this effect of the alcohol rather than to a 

 reaction between oxydase and benzidine. That this is so is indicated by the 

 fact that addition of hydrogen peroxide to a petal so treated causes a further 

 and immediate darkening. 



If, however, petals are immersed in absolute alcohol from which the 

 water has been removed by anhydrous copper sulphate, no browning occurs. 

 This is to be expected if the browning is due to oxydases, for, as shown in 

 Part IV, the oxydases are thrown out of action temporarily by dry alcohol. 



Petals transferred to water after soaking an hour or so in dry alcohol 

 rapidly develop the brown colour ; but petals that have been left several 

 days in the dry alcohol form no brown pigment on transference to water, 

 nor does the addition of hydrogen peroxide cause it to appear. If now it 

 be assumed that the formation of the brown pigment under the influence 

 of chloroform, alcohol, etc., is due to interaction between a colourless 

 " chromogen " and an oxydase (kept apart in the intact petal but allowed to 

 come together when the alcohol has destroyed the impermeability of the 

 plasmatic membrane) ; then the failure of the pigment to develop in the 

 case of petals that have been soaking some time in alcohol may be taken to 

 indicate that the chromogen has been removed from the petals and diffused 

 out into the alcohol. 



Failure of the brown colour to appear is not due to the destruction of the 

 body that functions as peroxide, since addition of hydrogen peroxide is 

 without effect ; nor is it due to the destruction of the peroxydase itself, 

 since the petal, after long immersion in alcohol, gives a good benzidine 

 reaction for peroxydase. 



If the above view of what occurs be correct, the absolute alcohol in which 

 the petals have been soaked should contain the chromogen in solution. In 

 order to prove that this is the case, a considerable number (150) of Lychnis 

 coronaria flowers were treated with 50-per-cent. alcohol, raised to boiling 

 point in order to destroy the oxydase. 



After concentrating the extract to a small bulk, white Lychnis flowers, 

 soaked in dry alcohol as above to remove the chromogen, and known to 

 contain peroxydase, were incubated at 36° C. in the solution. The flowers 

 remained colourless while in the extract, but when they were transferred to 

 water containing hydrogen peroxide a reddish-brown pigment appeared at 



2 A 2 



