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Porphyrin Detection in Denatured Cnidarian Tissue Extracts

Author(s): Avadanei, A.R.; L.A. May; C.M. Woodley

NCCOS Center: CCEHBR (http://coastalscience.noaa.gov/about/centers/ccehbr)

Publication Type: NOAA Technical Memoranda

Journal Title: NOAA Technical Memorandum NOS NCCOS 145 and CRCP 17

Date of Publication: 2012

Reference Information: 44 pages

Keywords: porphyrin; porphyrin detection; microplate fluorescence; denatured cnidarian tissue extracts; protoporphyrin IX; diagnostic; xenobiotic contaminants

Abstract: Porphyrin metabolic disruption from exposure to xenobiotic contaminants such as heavy metals, dioxins, and aromatic hydrocarbons can elicit overproduction of porphyrins. Measurement of porphyrin levels, when used in conjunction with other diagnostic assays, can help elucidate an organism’s physiological condition and provide evidence for exposure to certain toxicants. A sensitive microplate fluorometric assay has been optimized for detecting total porphyrin levels in detergent solubilized protein extracts from symbiotic, dinoflagellate-containing cnidarian tissues. The denaturing buffer used in this modified assay contains a number of potentially interfering components (e.g., sodium dodecyl sulfate (SDS), dithiothreitol (DTT), protease inhibitors, and chlorophyll from the symbiotic zooxanthellae), which required examination and validation. Examination of buffer components were validated for use in this porphyrin assay; while the use of a specific spectrofluorometric filter (excitation 400 ± 15 nm; emission 600 ± 20 nm) minimized chlorophyll interference. The detection limit for this assay is 10 fmol of total porphyrin per µg of total soluble protein and linearity is maintained up to 5000 fmol. The ability to measure total porphyrins in a SDS protein extract now allows a single extract to be used in multiple assays. This is an advantage over classical methods, particularly when tissue samples are limiting, as is often the case with coral due to availability and collection permit restrictions.

Availability: Cheryl.Woodley@noaa.gov

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