A MODIFIED MOLYBDENUM BLUE METHOD FOR ORTHOPHOSPHATE DETERMINATION SUITABLE FOR INVESTIGATING ENZYMATIC HYDROLYSIS OF ORGANIC PHOSPHATES

Authors: He, Zhongqi; Honeycutt, Charles

Submitted to: Communications in Soil Science and Plant Analysis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2004
Publication Date: 5/31/2005
Citation: He, Z., Honeycutt, C.W. 2005. A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates. Communications in Soil Science and Plant Analysis. Vol 36, pages 1373-1383.

Interpretive Summary: Phosphorus (P) is an essential nutrient for plant growth but also a potential pollutant contributing to eutrophication of surface waters. Investigation of the P forms and dynamics in environmental samples can provide insight in improving P management. Enzymatic hydrolysis is an approach to do this work. This approach requires a precise determination of the concentration of orthophosphate (Pi). In this study, we modified a molybdenum blue method to meet the requirement. This method modification provides increased sensitivity of Pi determination, thereby improving the accuracy and reliability of the enzymatic hydrolysis approach.

Technical Abstract: In characterizing organic P (P0) by phosphatase hydrolysis, the quantity of hydrolysable P0 is represented by the difference in orthophosphate (that is, inorganic P, Pi) determined after and prior to enzymatic incubation. Therefore, precise determination of Pi is of major importance for accurate application of the enzymatic hydrolysis approach. The strong acid conditions required for conventional molybdenum blue methods interfere with Pi determination due to rapid hydrolysis of labile P0 and precipitation of enzymes (proteins). The molybdenum blue method of Dick and Tabatabai (J. Environ. Qual. 1977, 6:82-85) reduces errors pertaining to non-enzymatic hydrolysis of P0. We revisited the method, finding that the absorption coefficient at 850 nm was 45-49% higher that at 700nm, and linear up to at least 80 nmol Pi in 1 mL assay solution. Therefore, adaptation of the readings at 850 nm improved the sensitivities of Pi determination by about 45%. Enzyme precipitation during Pi determination was prevented by addition of 2% SDS (sodium dodecyl sulfate) before color-forming reagents were added. This method modification provides increased sensitivity for Pi determination, thereby improving the accuracy of P0 analysis by phosphatase hydrolysis.