Measurement of enzyme kinetics in water samples: effect of freezing and soluble stabilizer

Abstract

The effect of freezing on the kinetics of enzymatic activity in water samples from both marine and limnic environments was tested in order to decide whether analysis of thawed samples could be used to estimate the magnitude of enzyme activity in samples. Samples stored at -20-degrees-C over 1 to 65 d were assayed using the fluorogenic substrates 4-methylumbelliferyl-beta-D-glucopyranoside (MUFGlp) and L-leucine-4-methyl-7-coumarinylamide (Leu-MCA). The establishment of enzyme-substrate saturation in thawed samples and the calculation of kinetic parameters for beta-D-glucosidase and leucine aminopeptidase showed: (1) a fast decrease in V(max) and (2) a fast increase of K(m) for seawater and freshwater samples. The observed decrease of V(max) and the loss in enzyme affinity led us to the conclusion that samples frozen at -20-degrees-C could not be used for an estimation of enzyme activity in the water samples. Attempts to stabilize the enzyme with 0.1% and 1% (v:v) glycerol at -20-degrees-C brought no significant improvement in obtaining higher enzyme activities. Also, freezing of the samples at -70-degrees-C or a combination of 10% glycerol and storage at -70-degrees-C gave no satisfactory results in the determination of enzyme activities in thawed samples. In searching for an alternative, an attempt was made to recover fluorescence by adding the fluorogenic substrate, and freezing the sample at -20-degrees-C after the incubation. The recovery of fluorescence (i.e. concentration of product of enzymatic reaction) after 1 and 10 d of storage was 100% for both beta-D-glucosidase and leucine aminopeptidase in seawater and freshwater samples. Fluorescence in water samples was not affected by repeated freezing and thawing processes. The method is recommended for storage of seawater and freshwater samples for enzyme activity measurement which cannot be immediately analyzed.