Measurement of Phytase Activity in a Clymer Forest Soil Using the TInsP5 Probe

Measurement of soil phytase activity (PA) and delineation of the impact of this important phosphomonoesterase on the P-cycling process in soil and sediments suffer from the lack of a reliable assay. A method for measuring PA in soil that promises to be accurate and reliable has been recently published. The method involves the use of a novel chromophoric analog of phytic acid, referred to as T(tethered)InsP5 (5-O-[6-(benzoylamino)hexyl]-D-myo-inositol-1,2,3,4,6-pentakisphosphate). This study was conducted to measure PA in a Clymer forest soil, which contained over twice the amount of soil organic C as previously tested soils, using the TInsP5 PA assay. This investigation specifically addresses: (1) the development of a soil dilution technique for determining maximal PA, (2) identification of previously unsubstantiated soil-produced dephosphorylated intermediate probe species, (3) the impact of increasing assay buffer pH on soil PA and (4) testing stability of the probe's amide bond in a highly (bio)active forest soil. PA assays were conducted by measuring dephosphorylation of TInsP5 in citrate-acetate buffered (pH 4.2) active and autoclaved (Control) soil suspensions. Phosphorylated probe intermediates (i.e., TInsP4, TInsP3, TInsP2 and TInsP1) and T-myo-inositol were extracted from samples of soil suspension following incubation. Probe species were quantified using reversed phase high-performance liquid chromatography (RPHPLC) with UV detection. PA was calculated based on a mass balance approach. A soil dilution technique was developed to address the challenge of determining maximal PA in soils containing higher organic matter content. In the initial report on use of the TInsP5 method for measuring PA in soil, two "soil-generated" UV-adsorbing compounds (designated Y and Z) were observed, but never confirmed as probe species. The experimental evidence presented in this report supports inclusion of compound Y as a phosphorylated probe intermediate species (i.e. TInsPy), based primarily on its UV adsorption spectra (diode-array detection analysis). Compound Z could not be substantiated as a probe species based on the evidence presented in this study. PA of Claymer forest soil decreased with an increase in assay buffer pH. Further, the probe's amide bond linkage was stable in a forest soil exhibiting high PA.