DIFFERENT OXIDANTS 1587 



may be essential for "energy clismutation/' e. g., via the formation of high 

 energy phosphate esters, as repeatedly discussed above) . 



No ox\'-gen liberation was observed in chloroplast suspensions with 

 glutathione (Holt and French 1948), or with dehydroascorbic acid (Aronoff 

 1946, Holt and French 1948), or with oxalacetic acid and riboflavin (Mehler 

 195P). (It will be recalled that the last named compound was used ex- 

 tensively by Krasnovsky for the dark reoxidation, in solution, of chloro- 

 phyll after its photoreduction by ascorbic acid, cf. part A of this chapter.) 



This may be the place to mention attempts to use other intermediary 

 metabolites, or miscellaneous biologically important substances, as Hill 

 oxidants. Several of these compounds (including phosphoglyceric and 

 pyruvic acid) were mentioned above as stimulating the photochemical oxy- 

 gen evolution mediated by the ''natural Hill oxidant" in crude leaf juices, 

 but causing no oxygen liberation with washed chloroplast fragments. 

 Another oxidant of the same type is methemoglobin. In studying the 

 "natural" Hill oxidant (first described in Hill's 1939 work, cf. page 63), 

 Davenport (1949) noted that the initial rate of methemoglobin reduction by 

 chloroplast suspensions was as high in the presence of a chloroplast free 

 leaf extract as in the presence of ferric oxalate. On the other hand, methe- 

 moglobin itself was not reduced photochemically by washed chloroplasts. 

 Following this lead, Davenport, Hill and Whatley (1952) found that crude 

 macerates of many leaves (cleared only by filtration through glass wool) re- 

 duced considerable amounts of methemoglobin in light. Results of this 

 type were obtained with leaves of Avena, Pisum sativu7n, Sambucus nigra, 

 Chenopodium Bonns-Henricus and Strellaria media. Similar preparations 

 from Calendula, Brassica and Tropaeolum were only weakly active, and 

 those from Phaseolus muUiflorus and Centranthus ruber were inactive. 



The activity (related to unit chlorophyll amount) decreased with dilu- 

 tion, e. g., from about 0.5 cm.* O2 per mg. chlorophyll per hour at [Chi] = 

 5.6 X 10-5 mole/1., to 0.15 cm. 3 at [Chi] = 1 X 10"^ mole/1. 



Figure 35.19F shows the loss of methemoglobin reducing capacity of 

 chloroplasts upon washing (by two centrifugations) and its restoration by 

 the addition of the supernatant from the first centrifugation, or of an aque- 

 ous extract from previously acetone-extracted leaf powder (prepared as 

 described on page 63). Similar aqueous extracts from acetone washed 

 root material, or from (chlorophyll free) terminal bud material, were inac- 

 tive. The bud extract inhibited the activity of the leaf extract, but the 

 root extract contained no such inhibiting components. The "methemo- 

 globin reducing" component of leaf extract was completely destroyed in 

 10 min. at 100° C, almost lost in 10 min. at 60° C, but remained almost 

 unaffected after 10 min. at 50° C. It was gradually deactivated by ex- 

 posure to air. It remained intact after overnight dialysis at 0° C, against 



