The chemistry of experimental chloroma. II. Isolation of crystalline protoporphyrin, its origin and relation to other porphyrins.

In a previous publication it was shown that the red fluorescence of chioroma tissue was due to porphyrins readily extracted with ethyl acetate and acetic acid ; and that a dicarboxylic acid porphyrin, probably protoporphyrin, was chiefly responsible for the fluorescence (9). The present report is concerned with the isolation of crystalline protoporphyrin, the quantitative comparison of the amounts of porphyrin in the â€oecoproporphyrin†• fractions, and â€oeprotoporphyrin†• fractions, the demonstration of the presence of at least thirteen chromatographic entities in these fractions, and the uptake of radioglycine-@-C'4 by the porphyrin pigments of chioroma tissue and the soft tissue of the animal body. The methodsof fractionationof the prophyrinsand their quantitative analysis are essentially those described by Schwartz and Wikoff for red blood cells (1@). The source of chioroma tissue (18) for the fractionation procedure was a pool of two chioromas weighing lfl gm.; four additional samples were used for quantitative comparison of amounts of porphyrins in the â€oeproto†• and â€oecopro†• fractions which were from the same animals used in the isotopic studies. In the latter, 1@ X 10' counts of glycine-%-C1' were injected subcu taneously into rats bearing chioromas which at autopsy @ and 45 hours later were found to weigh 9-10 gm. The handling of these tissues will be described under metabolic studies below. Preliminary fractionation.†" One hundred and seventeen gm. of chloroma tissue was homogenized with a 4:1 mixture of * This investigation was supported by a research grant ethyl acetate and acetic acid in a Waring Blendor. Filtration was carried out on a medium sintered glass filter and the residue further extracted until no fluorescence appeared in the filtrates. The ethyl acetate solution (A) was treated as follows: Uroporphyrins.†" Solution A was extracted with S per cent sodium acetate until no red fluorescent material could be re moved on further extraction. Chromatography on aluminum oxide columns, with ainmonium hydroxide as developing solvent, yielded a single, very slightly fluorescent band, which was not studied further. Coproporphyrins.†" Additional purification of the washed extract freed of acetic acid was achieved by passing the fluorescent material into 10 per cent HC1, neutralizing, and shifting the porphyrins back to ethyl acetate. The copropor phyrin fraction was then prepared by extracting with 1 per cent HC1, and was freed of contaminating porphyrins by reducing the HCI concentration to 0.4 per cent and extracting with chloroform. Forty-four pg. …