standardization of environmental data and information - International ...

3b
Parameter Variables Methodology for analyses
Sediment Phosphatase, lipase, protease and glucosidase
enzymes
Biochemistry
(cont.)
Sample preparation: Sediment from each subsection of core
air-dried, homogenized with mortar and pestle, and stored at
5? C pending analyses.
Method: Enzyme activity measured using substrate analogues
linked to fluorochrome methylumbelliferone (MUF) 8 with
sodium salt of MUF as standard; fluorescence intensity
measured and activity expressed as enzyme units (U) per g -1
of sediment per h -1 , where U = millimoles of MUF released.
Assay: Sediment slurry from each section diluted with
sterilized artificial seawater, homogenized and incubated in
centrifuge at 1 atmosphere in dark at room temperature
(28+2? C); reaction started by adding different concentration
of substrate (micromoles) in duplicate (each section’s
concentration of substrate varies); after incubation for 1 hr,
tubes centrifuged at 12,000 revolutions/minute at 4? C for
2' and relative fluorescence measured.
Phosphatase activity 9 :
Substrate: 4-MUF-phosphate
Standard: sodium salt of MUF
Fluorescence: excitation 365 nanometres, emission 455
nm
Lipase activity:
Substrate: MUF butyrate
Standard: sodium salt of MUF
Fluorescence: excitation 365 nm, emission 455 nm
Protease activity:
Substrate: leu-MCA (L-leucine-4-methylcoumarinyl-7-amide)
Standard: 7-amino-4-methylcoumarine
Fluorescence: excitation 380 nm, emission 440 nm
Glucosidase activity:
Substrate: MUF-?-d-glucosidase
Standard: Sodium salt of MUF
Fluorescence: excitation 365 nm, emission 455 nm
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INTERNATIONAL SEABED AUTHORITY