Do chromogenic assays of soil enzyme activities need buffers? More disadvantages than advantages of modified universal buffer in the para-nitrophenyl-based assay of phosphomonoesterase and β-glucosidase

Abstract

Buffers are commonly employed in soil enzyme assays to maintain a constant pH during the assay incubation, but soils are already buffered and buffer can alter apparent V_max and K_m. To test for potential artifacts of buffer on soil enzyme activities, we selected 32 soils to furnish a broad range of physiochemical characteristics and assayed soil β-glucosidase (BG) and phosphomonoesterase (PME) activities at varied substrate concentrations either in water or in modified universal buffer (MUB). The pH of assays was different (up to 1.6 units) from the measured soil pH (1:2, m/v in water), but MUB did not maintain pH better than water. Compared to water, MUB generally suppressed activities (by∼31%), apparent V_max (by∼32%) and K_m (by∼52%) of PME, but yielded similar activities (by∼4% difference) and apparent V_max (by∼9% difference) for BG. Soils with higher pH tended to have larger degree of suppressed PME actvities in MUB compared to assays in water. Based on the best practice of using a substrate concentration that is 5×K_m for substrate saturation of the enzyme, the median substrate requirement to assay PME in these 32 soils was ≈ 50 mM g^-1 in water and 25 mM g^-1 in MUB. Regardless of matrix type, the commonly employed PME substrate concentration of 10 mM g^-1 (e.g., Tabatabai, 1994) is insufficient for accurate activity assays. In contrast, for BG assays the commonly used pNP-linked substrate concentration of 10 mM g^-1 appears appropriate for most soils with a median substrate requirement of ∼4 mM g^-1 in water and ∼6 mM g^-1 in MUB. Our results support previous claims that buffers are unnecessary for assaying soil enzyme activities and can alter apparent kinetic parameters (K_m, V_max). Potential soil- and enzyme-specific substrate requirements should be determined a priori to ensure accurate measurements of enzyme activities in soils.