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SIMPLE METHOD FOR THE EXTRACTION OF PHOTOPIGMENTS AND MYCOSPORINE-LIKE AMINO ACIDS (MAAS) FROM SYMBIODINIUM SPP.
Citation:
Rogers, J E. AND D T. Marcovich. SIMPLE METHOD FOR THE EXTRACTION OF PHOTOPIGMENTS AND MYCOSPORINE-LIKE AMINO ACIDS (MAAS) FROM SYMBIODINIUM SPP. Presented at 7th International Conference on Coelenterate Biology, Lawrence, KS, Jul. 6-11, 2003.
Description:
Numerous extraction methods have been developed and used in the quantitation of both photopigments and mycosporine amino acids (MAAs) found in Symbiodinium sp. and zooanthellate metazoans. We have development of a simple, mild extraction procedure using methanol, which when coupled with HPLC-DAD analysis, quantifies the major photopigments and detects MAAs found in Symbiodinium sp. Wet cell pellets (approx. 2 x 107 cells) were suspended in 4.5 ml of methanol by vortex mixing. The mixture was placed on ice for 2 hr. after which 0.5 mL of 0.5 M ammonium acetate was added. After the solution was mixed by vortex and centrifuged to remove cell debris, a portion was removed and filtered (0.2 ?m PTFE filter membrane) into an amber HPLC vial. Complete extraction of both photopigments and putative MAAs was observed. A second extraction yielded only insignificant amounts of photopigments and MAAs. Samples were analyzed by the HPLC method of Wright et al. (Mar. Ecol. Prog. Ser. 77:183-196, 1991). Compounds were detected by diode array detection at 436 nm for photopigments and 334 nm for MAAs. MAAs were detected as a single irregular peak at 2.5 minutes which did not affect the quantification of photopigments which were first detected at 7.5 min (chlorophyll c). With another commonly used photopigment extraction method, sonication in acetone:water (90:10), recoveries of photopigments were only a fraction of those observed with methanol. For example acetone/water extract recovery compared to methanol was 72% for chlorophyll a, 74% for diadinoxanthin, 49% for peridinin, and 31% for chlorophyll c.