Formaldehyde preservation for deferred measurements of alkaline phosphatase activities in marine samples

Alkaline phosphatases are the main enzymes required by microorganisms to hydrolyse organic phosphorus into available phosphate in aquatic environments. The investigations of alkaline phosphatase activity (APA) usually generate numerous samples (size fractionation, Michaelis-Menten kinetics). Therefore, convenient and reliable preservation of incubated samples for a deferred analysis would be very useful when measurements cannot be performed right away. The APA of marine pond waters was measured using 4-Methylumbelliferyl phosphate (MUF-P) as the fluorogenic substrate modelling natural organic phosphorus compounds. Where typical inhibitors of other enzymatic activities, such as 1% sodium dodecyl sulfate, mercuric chloride, or buffered solutions of ammonium and glycine, failed to stop APA, the addition of formaldehyde efficiently inhibited APA. The effect of formaldehyde was the strongest with the highest concentration tested (4% final concentration) and in buffered (pH 8) solutions. Since a slow and gradual increase in APA may persist with time, the combination of the addition of 4% buffered formaldehyde with immediate freezing is the best method to entirely inhibit APA. The maximal rate of hydrolysis (V_max) and the Michaelis constant (K_m) of formaldehyde (4%)-inhibited samples did not significantly change during storage at -20 °C for 11 days. The method was successfully tested on samples with extremely high values of APA (15000-40000 nM h^-1) that were preserved for 1 month at -20 °C (98% inhibition). This method is a reliable and useful means of preserving incubated samples, and it provides convenient controls for background fluorescence of water and substrate, without provoking abiotic hydrolysis of the substrate.