1774 CHEMISTRY OF CHLOROPLAST PIGMENTS CHAP. 37B 



and could thus be presumed to be oxidation products. They were not 

 formed in the absence of air, or in dried leaves moistened with water and 

 then immersed into methanol (even in the presence of air). Formation 

 of the methyl chlorophyllides as well as of the colored oxidation products 

 could be prevented by preliminary exposure of leaves to boiling water 

 or steam, or to a temperature of 100° C. in a sealed tube — presumably 

 because heat destroyed l)oth the chlorophyllase and the enzymes catalyz- 

 ing oxidation. 



Sometimes pheophytins also were formed during the treatment of leaves 

 with methanol; this could be prevented by the addition of a base, such as 

 dimethylaniline. The formation of the chlorophyllides was faster when a 

 smaller volume of methanol was used, or aqueous methanol (50-75%) was 

 employed instead of pure solvent. 



In barley leaves, no chlorophyllase action occurred upon standing in 

 methanol ; if air was absent, they yielded mainly the original chlorophylls a 

 and b ; in air, the two above-mentioned, nonacidic oxidation products. The 

 oxidation enzymes resisted freezing but not dehydration. They were most 

 active in young seedlings. 



Strain analyzed products formed by standing in methanol also in leaves 

 of seventeen other species, and in isolated chloroplasts of Swiss chard. 

 Some gave mainly unaltered chlorophylls, and others chlorophyllides, 

 pheophytins or oxidized chlorophylls. 



A 10-20 hour treatment with acetone or methyl acetate caused chloro- 

 phyll in freshly chopped, or dried and moistened, mallow leaves to be con- 

 verted largely to chlorophyllin. Heating of dried leaves to 100° for 10 

 minutes did not prevent this conversion, but it could be avoided by a 1-2 

 minute immersion into boiling water. 



When chopped mallow leaves were permitted to stand in acetone in air 

 (but not in vacuum) for 24 hours, very strongly adsorbable pigments — 

 presumably oxidation products of the chlorophyllins — were formed. 



Barley leaves treated with acetone in air yielded primarily the same 

 oxidation products, which were also observed in treatment with alcohols. 



The green pigments formed from chlorophyll a by enzymatic oxidation 

 were found to differ from those formed by allomerization in solution. 

 Allomerized chlorophyll a is blue-green; absorption peaks lie {cf. fig. 

 21.4A) at 650 and 420 m/x (in methanol); on a sugar column, it forms a 

 band well above chlorophyll a. Xo trace of this band is visible in the chro- 

 matograph of enzymatic oxidation products from chopped barley leaves 

 left standing in methanol. On the other hand, the most strongly adsorbed 

 of the several minor green products formed in allomerization of chloro- 

 phyll a in methanol, appeared to be identical with the main enzymatic 

 oxidation product formed in leaves. Its band maximum lies at 662 m/x 



