A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates

In characterizing organic phosphorus (P-o) by phosphatase hydrolysis, the quantity of hydrolyzable P-o is represented by the difference in orthophosphate [i.e., inorganic P (P-i)] determined after and prior to enzymatic incubation. Therefore, precise determination of P-i is of major importance for accurate application of the enzymatic hydrolysis approach. The strong acid conditions required for conventional molybdenum blue methods interferes with P-i determination due to rapid hydrolysis of labile P-o and precipitation of enzymes (proteins). The molybdenum blue method of Dick and Tabatabai in 1977 reduced errors pertaining to nonenzymatic hydrolysis of P-o. This study revisited the method, finding that the absorption coefficient at 850 nm was 45-49% higher than at 700 nm, and linear up to at least 80 nmol P-i in 1-mL assay solution. Therefore, adaptation of the readings at 850 nm improved the sensitivities of P-i determination by about 45%. Enzyme precipitation during P-i determination was prevented by addition of 2% sodium dodecyl sulfate (SDS) before color-forming reagents were added. This method modification provides increased sensitivity for P-i determination, thereby improving the accuracy of P-o analysis by phosphatase hydrolysis.