Extracellular phosphatase enzyme activity in reef waters of the central Great Barrier Reef

Brief description

A fluorometric procedure was applied to measure changes in phosphatase activity in seawater flowing across reef flats at Bowl and Hopkinson Reefs on the central Great Barrier Reef. Seawater samples were collected at 50 m intervals across the reef flat during low tide, commencing at the reef front using a volume transport method and a time-distance-depth record was maintained allowing measurements to relate to the distance a water mass had flowed across the coral reef flat. Water samples were collected below the surface using a dip-scoop, filtered through 10 µm mesh and stored in the dark before being assayed.Fluorescence was measured on 2 ml filtered (Nucleopore, 0.2 µm) and unfiltered subsamples on addition of 50 µl of a 5.0 mM solution of the enzyme substrate (4-methylumbelliferyl phosphate) and then at hourly intervals. An 8 hour period of incubation was required to determine statistically significant rates of hydrolysis from oligotrophic water samples. This research was initiated to examine the importance of the hydrolysis of organophosphates by alkaline phosphatase enzymes as a source of inorganic phosphate, a nutrient essential for primary production. The assay used was able to determine low levels of enzyme phosphatase activity in marine waters based on the enzymatic hydrolysis of the fluorogenic substrate, 4-methylumbelliferyl phosphate.

Lineage

Maintenance and Update Frequency: notPlanned

Statement: Statement: The volume transport method used in collection of water samples is described in:Barnes DJ (1983) Profiling coral reef productivity and calcification using pH and oxygen electrodes. J. Exp. Mar. Biol. Ecol. 66: 149-169.Fluorescence was measured with a Turner spectrofluorometer, Model 430. Excitation wavelength was 364 nm (15 nm bandwidth) with a 7-60 band-pass filter (315-385 nm) to remove stray light artifacts. The emission wavelength was 458 nm (15 nm bandwidth) and a secondary 418 nm short wavelength cut-off filter was used to assist resolution of the 4-methylumbelliferone fluorescence from that of the unhydrolyzed enzyme substrate. Standard calibration was performed with 4- methylumbelliferone (Sigma Chemical) in filtered sterilized seawater (0.2 um). The calibration curve was linear between the intensity of 4- methylumbelliferone fluorescence and its concentration in the range 5 x 10-9 M to 2 x 10-6 M.Phosphatase enzyme activities were calculated using linear regression analyses and standard error determination provided values to calculate 95% confidence limits. Phosphatase enzyme activities are expressed in units of pmol substrate (or PO4) hydrolyzed/ml/h. Heat denatured sterile seawater (1 h at 100°C) was used for reagent controls. Blank values due to spontaneous, non-enzymatic hydrolysis of the substrate were substracted from the fluorescence assay as background (0.25 pmol substrate hydrolyzed/ml/h or lower). Spontaneous hydrolysis of the enzyme substrate was minimized by thorough cleaning of all glassware used in the analysis. Phosphate based detergents must be avoided as organophosphates are competitive substrates and orthophosphate inhibits the enzyme activity.

Notes

Credit
Dunlap, Walter C, Dr (Principal Investigator)