Alkalization solution affects selectivity of absorbance for colorimetric quantification of para-nitrophenol in soil enzyme activity assays

The alkalization step in soil enzyme activity assays can cause precipitation, potentially compromising colorimetric quantification of para-nitrophenol (pNP) product used to measure activity. We identified which soil enzyme activity assay matrices lead to the formation of a precipitate when alkalized using NaOH or tris(hydroxymethyl)aminomethane (Tris), characterized the precipitate, and quantified the extent to which the precipitate affected assayed soil enzyme activities. Precipitation occurred in matrices alkalized with 0.5 M NaOH (≈ 13 pH) but not in matrices alkalized with 0.1 M Tris (≈ 9 pH), and to a greater extent in matrices with modified universal (bufferuniversal) than acetate (bufferacetate) buffer. Thermodynamic modeling, mid-infrared Fourier transform infrared spectroscopy (FTIR), and elemental analysis indicated the precipitate was Ca(OH)2-based. Activities were higher for soil phosphomonoesterase assayed in bufferuniversal with NaOH than with Tris alkalization, but were similar for soil sulfatase assayed in bufferacetate. Alkalization with Tris avoids precipitates in soil enzyme activities and therefore stands to minimize inaccuracy of soil enzyme activity assays.