238 



Helen M. Habermann 



and allagochrome . 



Although we now have some working hypotheses concerning the 

 synthesis of allagochrome and evidence concerning its precursor, 

 the reality of the pigment as a component of the living cell re- 

 mains in doubt. Extraction at high pH , under oxidizing conditions 

 and in a buffer containing glycine provides all the necessary con- 

 ditions for production of an artifact. Attempts to find another 

 suitable buffer for extraction have been unsuccessful except for 

 borate-NaOH buffer. In this case, allagochrome values of sunflow- 

 er leaves were only 1/3 to 1/4 what they were with glycine-NaOH 

 buffer. It was not possible to increase yields in borate buffer 

 by adding glycine to the grinding medium. Although there are sev- 

 eral indications that at least part of the allagochrome found in 

 extracts of leaves exists in vivo, unequivocal proof of its natu- 

 ral occurrence is not yet available. 



CHARACTERIZATION OF ALLAGOCHROME 



Whether present in vivo or not, the pigment allagochrome re- 

 mains a chemically interesting molecule. The following paragraphs 

 summarize the kinds of information now available on which some 

 speculations concerning the configuration of allagochrome and of 

 the associated yellow pigments can be based. The latter pigments 

 are of increased interest at the present time because of their re- 

 semblance to phosphodoxin, a catalyst of photophosphorylation re- 

 cently reported by Black et al (^^ . 



Molecular weight 



A preliminary ultracentrifugal analysis was made with a 0.15% 

 solution of allagochrome in a Tris-HCl buffer (pH 8.2). Even pro- 

 longed centrifugation at maximum speed (59,780 rpm) did not result 

 in the formation of a boundary and the observed schlieren patterns 

 were characteristic of the transient states observed in an ap- 

 proach to equilibrium. Calculations of the molecular weight were 

 made by a method suggested by Ginsberg e_t aj. '-^^ and a value of 

 720 was obtained. The marked optical density of the solution lim- 

 ited the accuracy attainable by this method. The value should 

 therefore be considered a lower limit and the true molecular 

 weight probably lies between 720 and 1400. This method of analy- 

 sis gave no information about the homogeneity of the sample but 

 did indicate that no protein was present because of the absence 

 of high molecular weight components. 



Electron spin resonance 



The first ESR spectra of solid allagochrome and a frozen water 

 solution (containing 2 mg/ml) gave a 12 gaus peak to peak signal 

 at g=2.005. This was the only signal found at that time and on 

 this basis it was concluded that the common paramagnetic metal 



