Coupled, continuous and discontinuous fluorometric assays for trehalase activity

Abstract

Continuous and discontinuous coupled fluorometric assays which couple trehalose hydrolysis to peroxidation of the fluorogenic compounds eugenol (4-allyl-2-methoxyphenol) or p-hydroxyphenylacetic acid using glucose oxidase (EC 1.1.3.4) and peroxidase (EC 1.11.1.7) as ancillary enzymes have been developed for the measurement of trehalase (α,α′-trehalose-1- d-glucohydrolase, EC 3.2.1.28) activity from the cellular slime mold, Dictyostelium discoideum. With these methods, product formation was linear with time and the coupled reaction rate was directly proportional to the level of enzyme assayed. The validity of both the discontinuous and continuous fluorometric assays was confirmed by comparative studies with discontinuous spectrophotometric assays for glucose. Levels of glucose as low as 0.02 nmol were measurable with the discontinuous fluorometric procedures, thereby making the latter about 500-fold more sensitive than routine spectrophotometric assays. With the continuous fluorometric trehalase assays, the lower limits of sensitivity correspond to enzyme levels of the order of 5 to 25 μunits. The high level of sensitivity achieved with these assays makes them ideally well suited for: (i) elucidation of the regulatory mechanisms underlying the dramatic changes in trehalase activity that occur during spore germination and cellular aggregation in Dictyostelium and (ii) characterization studies involving electrophoresis or isoelectric focusing of trehalase in solid matrices in which enzymatic activity is measured either quantitatively in gel eluates or qualitatively by the in situ localization of the enzyme histochemically.