Dataset: SO248: GF MCAMUF hydrolysis rates
Deployment: SO248

Water was collected via Niskin bottles mounted on a rosette, equipped with a CTD.

Experiments on (operationally defined) particles were carried out by gravity-filtering water through 3 µm pore size filters.  1/12th sections of the 3 µm pore-size filters were submerged in 4 mL artificial seawater in incubation cuvettes.  Particle-associated peptidase and glucosidase activity assays were set up in 4 mL cuvettes. For live duplicate incubations, two particle-containing filter pieces (each 1/12th of entire filter) were separately submerged in 4 mL of cooled, autoclaved ambient seawater. A single killed control was prepared by submerging a sterile filter piece (1/12th of unused filter) in 4 mL of cooled, autoclaved ambient seawater. Substrates were added to a final concentration of 100 µM. At various timepoints—upon addition of substrate (to), 24 h (t1), 48 (t2), and 72 h (t3)—live duplicates and killed control singleton were subsampled by taking 3 x 200 uL (for technical triplicates) per incubation, and placed in a 96 well plate for fluorescence measurement using the Tecan Plate Reader. Fluorescence values were converted to hydrolysis rates using calibration curves with the MCA and MUF fluorophores, and were  normalized by the volume of filtrate that passed through the 3 µm filter. Two substrates, -glucose and -glucose linked to a 4-methylumbelliferyl (MUF) fluorophore, were used to measure glucosidase activities. 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 phenylalanine-serine-arginine (FSR) – 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].

a-glu = substrate to measure alpha glucosidase: 4-methylumbelliferyl-a-D-glucopyranoside
b-glu = substrate to measure beta glucosidase: 4-methylumbelliferyl-ß-D-glucopyranoside
L = substrate to measure leucine aminopeptidase (L-leucine-7-amido-4 MCA)
AAF = substrate to measure chymotrypsin activity: ala-ala-phe-MCA
AAPF = substrate to measure chymotrypsin activity: N-succinyl-ala-ala-pro-phe-MCA
QAR = substrate to measure trypsin activity: Boc-gln-ala-arg-MCA  
FSR = substrate to measure trypsin activity: N-t-boc-phe-ser-arg-MCA