Abstract— The small amounts of chlorophyll precursors in dark-grown cells of Euglena gracilis Klebs var. bacillaris Pringsheim and the presence of contaminants which interfere with their purification have made their isolation and characterization difficult. We now extract cells with acetone: 0.1 M NH4OH (9:1 v/v). Protochlorophyll is obtained by extracting this solution with petroleum ether (30–50° b.p.) and extracting this petroleum ether fraction with 80% acetone to remove substances which interfere with subsequent chromatography. Protochlorophyllide remaining in the original acetone: NH4OH fraction is extractable into diethyl ether after adjusting the pH to approximately 5.5. Both pigments are verified and further purified by chromatography on Whatmann 3 MM paper using benzene:petroleum ether:acetone (30:10:1) or cellulose MN 300 thin layers with methanol:methylene chloride:water (100:18:20). These pigments resemble their well-described barley counterparts in solubility properties, spectral absorption maxima in ether (432, 530, 571 and 623 nm) and chromatographic behavior. Substantial amounts of protopheophytin and protopheophoribide are also found along with the Mg2+— containing pigments in cell extracts even when precautions are taken to avoid loss of Mg2+ during extractions and purification.
Using heated cells before and chilled solvents after illumination to preclude enzymatic modification of pigments, chlorophyll and very small amounts of chlorophyllide are found as products of the photo-conversion of the protopigments suggesting that both protochlorophyll and, to a much smaller extent, protochlorophyllide are photoconvertible in these organisms. These properties join several others which suggest that the Euglena chorophyll-forming system more closely resembles that found in young bean leaves rather than the chlorophyll-forming system of the older leaf material usually studied.