An HPLC-based assay for soil enzyme activities using 4-nitrophenyl substrates

Soil enzyme activity assays are useful for assessing the biogeochemical potential of soils. Frequently, 4-nitrophenyl-labelled chromogenic substrates are used that yield yellow 4-nitrophenol (4-NP), which can be quantified by spectrophotometry at 410 nm. However, this approach is often impeded by the presence of organic matter, which causes yellow or even brownish colouring of the soil extracts, interfering with spectrophotometric measurements. In this study, we show that high-performance liquid chromatography (HPLC) is an efficient, rapid and selective method for quantifying the liberated 4-NP. The soil samples were mixed with the substrates dissolved in buffers and incubated for a suitable time period. Subsequently, the reactions were analysed by HPLC using a LiChroCART RP18 3 µm 55 × 2 mm with an eluent consisting of 20 mM acetic acid in acetonitrile/water = 30/70 (v/v) at a flow rate of 0.4 mL/min, a run time of 2.7 min and absorbance detection at 320 nm. This method is particularly suitable for carbon-rich soils, omitting the need for substrate-free controls, and offers excellent reproducibility while maintaining the simplicity and accuracy of traditional soil enzyme assays. • Simple, selective and highly reproducible method for measurement of soil enzyme activities. • Application of 4-nitrophenyl substrates. • Efficient separation of 4-nitrophenol from humic acids.