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Helen M. Habermann 



the minimum in difference spectra. Chlorogenic acid extracted 

 from leaves with boiling water is just as effective in increasing 

 yields as the commercially available chemical. 



3) The participation of polyphenol oxidase in formation of colored 

 derivatives of chlorogenic acid (especially during aging or after 

 injury of leaves) has been proposed by a number of investigators 

 (8,9)^ The rapid secondary synthesis of excess allagochrome from 

 chlorogenic acid added during grinding suggested that an enzyme of 

 this type is present and could account for part or all of the pig- 

 ment found in leaf extracts. The extent of enzymatic formation of 

 allagochrome during grinding was estimated in two ways: by adding 

 cyanide to the extracting medium or by heating leaves in boiling 

 water before extraction. Cyanide added to the extracting buffer 

 reduces allagochrome values (see fig. 1). Heating leaves before 

 extraction similarly reduces allagochrome values in controls and 

 inhibits the secondary synthesis of pigment from added chlorogenic 

 acid. Immersing leaves in boiling water for as short a time as 5 

 sec. reduces allagochrome values to about half, but longer heating 

 results in proportionately less additional reduction of pigment 

 values of the extracts (see fig. 2). Reduction in allagochrome 

 values is not proportional to removal of chlorogenic acid. Less 

 than 17o is removed by 5 sec. in boiling water and maximum extrac- 

 tion is achieved by this means in 1 1/2 to 2 min . In experiments 

 testing the effects of heating on the secondary synthesis of al- 

 lagochrome from added chlorogenic acid, only 30 sec. heating be- 

 fore grinding reduced the secondary synthesis to zero; shorter 

 heating resulted in reduction of the secondary synthesis in pro- 

 portion to the time of treatment over the entire range of added 

 amounts of chlorogenic acid (see fig. 3). 



4) Plants testing negatively for allagochrome respond in two pos- 

 sible ways when chlorogenic acid is added during grinding: they 

 either remain negative or become positive. This may be inter- 

 preted as an allagochrome negative, enzyme negative vs. allago- 

 chrome negative, enzyme positive situation; or it may indicate the 

 presence of a natural antioxidant which interferes with quinone 

 formation. We have not yet resolved this question. 



It seems quite probable then that the synthesis of allagochrome 

 from chlorogenic acid and glycine is mediated by an enzyme of the 

 polyphenol oxidase type. This has been substantiated by experi- 

 ments in which sunflowers were grown on copper deficient nutrient 

 solutions (7) . Such plants showed progressively lower copper con- 

 tent of leaves formed at successive nodes and markedly decreased 

 allagochrome levels. At the higher nodes, allagochrome values 

 dropped to between 10 and 207, of controls. There was an increased 

 susceptibility to cyanide poisoning of respiratory oxygen uptake 

 and photosynthetic oxygen production in leaves deficient in copper 



