Water was collected via Niskin bottles mounted on a rosette, equipped with a CTD.
Five substrates linked to a 7-amido-4-methyl coumarin (MCA) fluorophore, one amino acid – leucine – and four oligopeptides – the chymotrypsin substrates alanine-alanine-phenylalanine (AAF) and alanine-alanine-proline-phenylalanine (AAPF), and the trypsin substrates glutamine-alanine-arginine (QAR) and glutamic acid-gylcine-arginine (EGR) – were used to measure exo- and endo-acting peptidase activities, respectively. Hydrolysis rates of the substrates were measured as an increase in fluorescence as the fluorophore was hydrolyzed from the substrate over time [as in Hoppe, 1993; Obayashi and Suzuki, 2005]. Incubations with the seven low molecular weight substrates were set up in 4 ml cuvettes. For each substrate, triplicate cuvettes were filled with a total volume of 4 ml seawater for experimental incubations; single cuvettes were filled with 4 ml autoclaved seawater for killed control incubations. Substrate was added 100 micromolar concentrations. Fluorescence was measured over at 0 hrs, 24 h, 48h, and 72 h, using a mini fluorimeter. Hydrolysis rates were calculated from the rate of increase of fluorescence in the incubation over time relative to a set of standards of known concentration of fluorophore.
All incubations were conducted at 0 C in the dark.
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