270 PORPHYRINS 



COMPOUND HCl NUMBER 

 Coproporphyrin III 0. 09 



Protoporphyrin IX 2.0 



Pheophytin a 29.0 



A larger compilation of values is given by Granick and Gilder (27). 



Paper chromatographic procedures have not been applied to the porphyrins as ex- 

 tensively as to some other classes of compounds. Separation of metal-free porphyrins 

 has been achieved by Nicholas (28) and Kehl and Stich (29) with a lutidine -water system, 

 but closely related isomers cannot be separated in this way. By converting porphyrins 

 to their methyl esters and using solvent mixtures of chloroform, kerosene, propanol and 

 dioxane, Chu, Green and Chu (30) were able to separate closely allied compounds (cf. 

 also 31). Paper chromatography of the pigments related to chlorophyll has been devel- 

 oped by several workers (32-36). Non-polar solvents such as petroleum ether, toluene, 

 chlorobenzene, with sometimes a trace of an alcohol added, have been most effective. 

 Spots on the chromatograms are easily detected, even at minute concentrations, because 

 of their intense fluorescence in ultraviolet light. No attempt seems to have been made 

 to apply paper chromatography to the iron porphyrins. 



Preliminary structure determinations on the porphyrins should be facilitated by the 

 procedure of Nicolaus et al. (37, 38, 39) whereby small quantities of porphyrin can be ox- 

 idized with alkaline permanganate and the split products identified on paper chromato- 

 grams. These products are pyrrole acids and can be detected by spraying with diazotized 

 sulfanilic acid. The acids obtained indicate what side chains are present but not, of course, 

 the order of the pyrrole rings around the porphyrin nucleus. 



METABOLIC PATHWAYS 



The accompanying chart shows the probable course of biosynthesis for both the 

 hematin and chlorophyll pigments in higher plants. 



Biosynthesis of porphyrins (including chlorophyll) has been reviewed by Gibson 

 et aZ.(40). Several of the enzymes catalyzing reactions of porphyrin synthesis have been 

 demonstrated in wheat leaves (41); but, in general, the porphyrin biosynthetic pathway 

 has been studied very little in the higher plants. The first stages from succinate plus 

 glycine to protoporphyrin have been primarily investigated in vertebrate tissues. The 

 later stages in chlorophyll formation have been developed by studies on algae (42, 43). 

 The conversion of protochlorophyll to chlorophyll has been well-substantiated in higher 

 plants although there may be differences in detail from plant to plant. In some, phytol 

 may be already esterified to the protochlorophyll so that a single reductive step forms 

 chlorophyll. In others protochlorophyll may have a free carboxyl group which is esteri- 

 fied with phytol after ring IV has been reduced (44-48). There is also some question 

 about whether a single protochlorophyll is present which forms chlorophyll a, followed 

 by conversion of a into b or whether there may be separate protochlorophylls a and b 

 (49, 50). Bogorad (51, 52) has shown that spinach leaf preparations can catalyze the for- 

 mation of uroporphyrin from porphobilinogen; and Goodwin et al. (53) have identified a 

 compound like uroporphyrin as a normal constituent of certain Vicia cells. 



The overall reaction: 



6 - aminolevulinic acid — chlorophylls 



has been studied by Duranton et al. (54) who fed 6 - aminolevulinic acid-4-C" to tobacco 

 plants. In the dark radioactivity appeared in protochlorophyll, in the light in chlorophylls 

 a and b with three times higher activity in the a form. Similar results have also been 



