THE PATHWAY OF METMYOGLOBIN AND NADP REDUCTION 

 BY ILLUMINATED CHLOROPLASTS 



H. E. Davenport 



It has been shown^l) that the "methaemoglobin reducing factor" of Daven- 

 port, Hill and Whatley(2) is in its purified form^^) identical with the "photo- 

 synthetic pyridine nucleotide reductase" (PPNR) of San Pietro and Lang(4). 

 More recently the name ferredoxin^^^ has been proposed for this same leaf 

 protein and will be adopted in what follows. 



When a comparison was made of the catalytic activities of purified 

 preparations of ferredoxin in promoting the photochemical reduction of nicotin- 

 amide adenine dinucleotide phosphate (NADP) on the one hand, and sperm whale 

 metmyoglobin on the other, it was found that the relative rates of these two 

 reactions, measured under identical conditions, may show wide variations. 

 The variability did not appear to be related to the source or method of prepara- 

 tion of the ferredoxin but rather to the source and pretreatment of the chloro- 

 plasts or grana used in the assay mixtures. The garden pea (Pisum sativum ) 

 yielded chloroplast material showing the widest variability, and spinach 

 (Spinacea oleracea) the least. Pea chloroplast material was therefore chosen 

 for use in a systematic examination of the causes of the observed variability. 



It had been shown earlier^!' 2, 3) that when pea chloroplasts are prepared 

 in isotonic sucrose a single further wash in the same medium suffices to abolish 

 their capacity to reduce NADP or metmyoglobin unless ferredoxin is added 

 back to the reaction mixture. For this reason it was not possible to establish 

 conclusively that ferredoxin is located exclusively in the chloroplast. When 

 such ferredoxin-depleted pea chloroplasts are fragmented in dilute buffer and 

 washed repeatedly in water the pattern of their reactivity towards NADP and 

 metmyoglobin, assayed in the presence of adequate ferredoxin, undergoes a 

 progressive change. In the experiment cited in Table 1 the capacity of the 

 fragments to reduce NADP was diminished by 87% after six washes whereas 

 the reduction rate with metmyoglobin increased by 60%. In attempts to reverse 

 the inactivation of NADP reduction the pooled wash-fluid was subjected to frac- 

 tionation with ammonium sulphate. A protein fraction, precipitated between 

 the limits 50-66% saturation was found, after prolonged dialysis to remove 

 ammonium sulphate, to be capable of restoring completely the NADP reducing 

 activity of the chloroplast fragments. The addition of this fraction to the assay 

 system was without effect on metmyoglobin reduction. 



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