The use of the curves produced over the total incubation period is necessary to

observe enzyme behavior over time. While the endpoint based activity (Tf-Ti) produced

as a result of incubation may indicate a net change in substrate converted, there is often

erratic conversion behavior over time. Without the use of these curves, the data would be

considered valid since there would be no reason to discard any values that appear to

reflect net activity. The resultant experimental protocol for future studies discards any

non-linear runs from further analysis when the activity reflects outlying values.

 In determining optimum substrate concentrations, it is generally evident that very

high and low concentrations tended to produce erratic changes in activity over time and

lower differences between sites. The high concentration response can be attributed to

intermolecular quenching in which fluorescence is masked by the presence of other

fluorescent and non-fluorescent molecules. Therefore, there can be a false decrease or a

lesser increase in substrate conversion as measured by the fluorimeter. At low

concentrations, saturation kinetics did not appear to be satisfied, thereby resulting in a

lower perceived reaction rates. This is due to the lack of adequate substrate that fails to

retain a constant reaction velocity. The appropriate MUF and AMC substrate

concentrations that were determined in this study fall within the range of 40 to 200 [tM

documented in other studies of water column and soil enzymes (Bums and Ryder, 2001;

Shackle et al., 2000; Sinsabaugh and Foreman, 2001; Saiya-Cork et al., 2002).

 Incubation time was shown to influence the resolution between the two study

sites. In most cases, incubation times less than 30 minutes result in a decrease in

resolution. In addition, substrate conversion is generally erratic with large errors between

replicates. An incubation time of 45 minutes is deemed suitable for capturing activity as