MEPS 277:285-290 (2004)  -  doi:10.3354/meps277285

ABSTRACT: Measurements of potential enzyme activities are usually carried out to compare enzymatic activity between different systems and minimise the effect of product inhibition or substrate depletion during the assay. There is a general practice of estimating potential alkaline phosphatase activity (APA) using single-substrate addition in excess of saturation, as complete kinetic experiments are time-consuming. Here, complete APA kinetic experiments were carried out in the Subtropical North Atlantic Ocean using from 0.1 to 100 µM concentration of the fluorogenic substrate MUF-P. There was a reduction of the velocity of reaction by excess of substrate in all experiments carried out (n = 7). This reduction could be related either to inhibition processes, to quenching effects, or to a loss of fluorescence of the product of reaction due to some kind of biological transformation. Moreover, the substrate concentration above which activity diminished was different between the cases studied. Therefore, there was only a narrow range of concentration in which single-substrate addition experiments would have given results comparable to the maximum enzyme activity, and this range differed between stations. Hence, in order to estimate APA potential activities in marine oligotrophic environments, complete kinetic experiments should be carried out, since the use of single additions in excess of substrate may give erroneous results. This conclusion may be extendable to other commonly performed assays of hydrolytic activities in aquatic environments that also use MUF-substrates.

KEY WORDS: Alkaline phosphatase activity · Exoenzyme · Potential assays · Marine environments