Microplate fluorimetric assay of soil leucine aminopeptidase activity: alkalization is not needed before fluorescence reading

Abstract

The fluorescence responses of 7-amino-4-methylcoumarin (AMC) and L-leucine-7-amido-4-methylcoumarin (L-leucine-AMC) (the fluorescent dye and substrate used in the soil leucine aminopeptidase assay, respectively) to alkalization by NaOH and tris(hydroxymethyl)aminomethane (THAM) buffer were analyzed. The leucine aminopeptidase activities were also measured without and with the alkalization procedure using five soils with contrasting properties. The pH optimum of leucine aminopeptidase activity was 8 ± 1 for the five studied soils. In this pH range, the alkalization procedure cannot increase the fluorescence of AMC to improve the assay sensitivity. Excess alkalization (final medium pH of 12 or higher) led to decreasing fluorescence of AMC with time and non-enzymatic hydrolysis of L-leucine-AMC, which caused irreproducible values of the calculated enzyme activities. Moreover, a medium pH of ~ 11 cannot terminate the enzymatic reaction, and thus it seems impractical to stop the enzyme reaction by alkalization without causing the unstable behavior of AMC and L-leucine-AMC. We recommend performing the soil leucine aminopeptidase assay at the optimal pH without alkalization and reading the fluorescence immediately after incubation. This approach may help to simplify and standardize the leucine aminopeptidase assay.