501 



Joseph S. Kahn 



Samples of the extract were dried, dissolved in n-heptane 

 and chromatographed on aluminum oxide, powdered sugar, and 

 starch by thin- layer chromatography. On the basis of the 

 absorption spectra and the chromatographic behavior the pig- 

 ment was identified as a carotenoid, probably a carotene. 



The carotene-containing extract was further purified by 

 chromatography on DEAE-cellulose , all the carotene appearing in 

 a single peak containing 8.6-10.8 mg protein/^mole carotene. 

 This protein was firmly bound to the carotene and could be 

 separated only in part by extraction with dry solvents. 



The molar ratio of chlorophyll to carotene in the crude 

 protein-chlorophyll varied from 7 to 14. The protein- 

 chlorophyll from which the protein-carotene had been extracted 

 lost its ability to reduce ferricyanide, but the activity could 

 be restored by adding back the protein-carotene, the optimal 

 molar ratio of chlorophyll to carotene being about 10. When 

 added to whole or broken chloroplasts , the protein-carotene 

 completely inhibited ferricyanide reduction. Plastoquinone, 

 y?-carotene and FMN were unable to reactivate the protein- 

 chlorophyll, but menadione could substitute for the protein- 

 carotene. The optimal concentration for menadione was, however, 

 1000 times higher than that of the protein-carotene. The data 

 are summarized in Table IV. This effect of menadione will 

 explain its consistent small stimulation of ferricyanide re- 

 duction in crude protein-chlorophyll (Table I), since most 

 preparations had less than the optimal concentration of protein- 

 carotene . 



The protein-carotene was not changed during ferricyanide re- 

 duction, and could be recovered intact after total bleaching of 

 the chlorophyll. No stoichiometry existed between the amount of 

 protein-carotene present and the amount of chlorophyll bleached. 



In conclusion, we have isolated soluble protein-chlorophyll 

 and protein-carotene complexes from spinach chloroplasts which 

 together are able to photoreduce ferricyanide. In light of the 

 fact that no oxygen was evolved during the reduction of ferri- 

 cyanide, and that CMU had no inhibitory effect, these complexes 

 may represent a model for that photochemical reaction in photo- 

 synthesis, involved in raising the potential of the reductant 

 enough to reduce NADP. 



The protein moiety in both complexes appears to have a non- 

 enzymatic function in their activity, and may be the factor 



