Sources of abiotic hydrolysis of chromogenic substrates in soil enzyme assays: Storage, termination, and incubation

Assays of enzyme activities using chromogenic substrates that release para-nitrophenol (pNP) and para-nitroaniline (pNA) products are commonly employed in soil science, but these substrates are susceptible to non-enzymatic (i.e., abiotic) hydrolysis. We evaluated abiotic hydrolysis of 10 pNP- and pNA-linked substrates stored over seven days in two matrices of water and modified universal buffer, and with two alkalization methods of 0.5 M NaOH and 0.1 M Tris. We then compared the magnitude of abiotic versus enzymatic hydrolysis of substrates for two soils with high and low enzyme activity. Finally, we quantified substrate abiotic hydrolysis during the incubation (1–2 h at 37 °C). Abiotic hydrolysis of stored substrate solutions remained relatively constant across 7 days, and the base type used in alkalization had a much stronger effect on abiotic hydrolysis than storage time or matrix. Abiotic hydrolysis was generally least for substrates dissolved in water with Tris alkalization and greatest when dissolved in modified universal buffer with NaOH alkalization. The extent of abiotic hydrolysis varied by substrate, and in general was least for ester substrates and greatest for amide substrates. Abiotic hydrolysis was as low as <0.7% for the glycosidic substrate used to assay β-N-acetyl-glucosaminidase, and as high as 52–57% for amide substrates used to assay aminopeptidases. The magnitude of abiotic hydrolysis was more appreciable, and in some cases greater, than total substrate hydrolysis for the soil with overall low enzyme activities. Finally, appreciable abiotic hydrolysis occurred during the incubation, indicating that the commonly employed control for non-enzymatic pNP or pNA products in which substrate solution is added to a soil after the assay incubation is not appropriate. In order to minimize abiotic hydrolysis, we recommend alkalization of assays using 0.1 M Tris instead of 0.5 M NaOH; appreciable decreases in abiotic hydrolysis can also be achieved for water-only assays. To accurately control for abiotic hydrolysis in soil enzyme assays, incubated soil-free substrate-only controls should be used.