Chlorophyllase and Formation of an Atypical Chlorophyllide in Marine Algae

Abstract

In the course of a study of the pigment composition of some marine algae (6) certain organisms were found to possess highly active systems capable of converting chlorophyll a to chlorophyllide derivatives. Two chlorophyllides could be obtained from the diatom Phaeodactylnin tricornutunt (Bohlin) depending on the conditions of incubation. One possessed a spectrum identical with chlorophyll a, and therefore resembled the normal chlorophyllide a of higher plants (5) ; the other had a spectrum which differed from that of chlorophyll a in the Soret band region and was shown to be an atypical chlorophyllide, having a hydroxyl group of either alcohol or enol type. The present work is concerned with the properties of the chlorophyllides from Phaeodactvlumt tricornutnim, and the distribution of the chlorophyllase among some marine algae. Materials and Methods Marine Algae. The marine organisms used (see table II) were grown in the Erdschreiber medium described previously (6). The diatom Phaeodactylum tricornutum was used as the source of the chlorophyl-lides which were examined chemically and spectropho-tometrically. Cells were harvested after 1 to 2 weeks of growth by centrifuging at 2,000 X g for 5 minutes. Chlorophyllase Assay. Chlorophyllase activity was tested by suspending the harvested algae in 50% acetone and incubating at 20° for 15 minutes. After this period, acetone was added to a final concentration of 90 to 95% to inactivate the enzyme, and the cell residue was removed by centrifugation. The residue was reextracted with 100% acetone until colorless. The pigments in the combined acetone extracts were transferred from acetone to ethyl ether by mixing the acetone extract with an equal volume of ethyl ether, and shaking the mixture with 5 times its volume of 10% sodium chloride solution. The ether solution containing the pigments was then concentrated for chromatography. Paper Chromnatographly of Chlorophyll a and Chlorophyllides. The extract was applied to What-man No. 3 MM papers and chromatographed with 4% n-propanol in light petroleum (60 80°) in the dark at room temperature. Chlorophyll a and the chlorophyllides had RF, values of 0.6 and 0.2 respec-1 44 tively in this solvent. After development of the chromatogram the chlorophyll a and chlorophyllide spots were cut out, eluted with acetone, and the ab-sorbance read at 665 mn in a Unicam SP 500 spectro-photometer. Chlorophyll conversion was expressed as the percentage of chlorophyllide a formed from chlorophyll a in 15 minutes in 50% acetone at 20°. The extinction coefficient of the chlorophyllide was assumed to be identical with that of chlorophyll a. For the preparation of chlorophyllide from Phaeod-actylumin tricornu turn ior chemical and spectrophoto-metric characterization, cells were incubated in 50% acetone for 15 minutes at 20°. The pigments were then extracted and transferred to ethyl ether as above. The chlorophyllide was separated from the other pigments by chromatographing the extracts on paper, first with ethyl ether and then with chloroform in the same direction. In ethyl ether, carotene, chlorophyll a, and xanthophylls travelled with the solvent front (RF 0.95) followed by the chlorophyllide (RF 0.6) with chlorophyll c just above the origin (RF 0.2). The second development with chloroform washed traces of fucoxanthin from the chlorophyllide zone, with only slight increases in the RF values of the chlorophyllide and chlorophyll c. The chlorophyllide was eluted from the paper with acetone and stored in the dark at-20°. Absorbance measurements were made immediately after preparation of the chlorophyl-lide by transferring a portion of the pigment to ethyl ether as described above. The absorption curves were obtained using a Carv recording spectrophotometer (model 14). Solvents. Chromatographic solvents used were analytical reagent grade and were not further purified. Acetone was a commercial grade and was purified by storage over K2CO0, followed by distillation. Results Conzversion, of Chlorophyll a to Chllorophlvllide. The properties of the system wsvhich indicated that the conversion of chlorophyll a to chlorophyllide was an enzymic change are shown in table I, using Phaeod-actylutn, tricornutuim as the test organism. Maximal activity (90% conversion in 5 min) took place in 50 to 60%' acetone with almost complete inhibition of the process in 80 to 100% acetone. Heat inactivated cells showed little or no chlorophyllide formation when incubated with 50% acetone, and no chlorophyllide was obtained from cells which were extracted from the frozen state at-10 to-20° in 100% acetone.