We report below on an extremely simple and somewhat more sensitive modification of the Chert procedure (1) for inorganic orthophosphate determination that has been routinely used in this laboratory for almost 2 years. The method overcomes the problem of precipitation of the phosphomolybdate complex in the presence of Triton X-100, a detergent commonly employed in assays of membrane-bound phosphohydrolases. We have utilized the original observation of See and Fitt (2) regarding the solubility of the precipitated phosphomolybdate complex if the concentration of Triton X-100 is increased sufficiently.
The following stock solutions were prepared: (a) 2 N H2SO4 containing 2.0% v/v Triton X-100; (b) 2.5% aqueous ammonium molybdate; and (c) 10.0% aqueous ascorbic acid. Reagent c was prepared fresh each week and stored at 4ยฐC, the other stock solutions were stored at room temperature in plastic bottles and were good for several weeks.
To determine liberated orthophosphate in enzyme assay mixtures (generally containing 0.075-0.10% Triton X-100), reactions were terminated after a suitable incubation using 0.5 vol of 0.6 N perchloric acid. The samples were then diluted with distilled water lowering the perchloric acid concentration to 0.075 y. The molybdate reagent was then prepared by mixing 3 vol (a), 1 vol (b), and 1 vol (c), and an equal volume of this reagent added to each diluted sample. After a 15-min incubation at 30ยฐC, the absorbance was read at 790 nm using a Unican SP1800 double-beam spectrophotometer with an appropriate zero time blank in the reference cell compartment. In most instances, we removed precipitated protein by centrifugation at 10,000g for 10 rain prior to addition of the molybdate reagent. Unreacted labile substrates, i.e., nucleoside phosphates, were removed with Darco G-60 activated charcoal (10.0 g charcoal/100 ml 0.6 N perchloric acid), centrifuging down the charcoal along with the precipitated protein.
A wavelength scan between 710-840 nm revealed enhanced absorption by the reduced phosphomolybdate complex in the presence of Triton X-100. Moreover maximum absorption in the presence of detergent occurred at a wavelength of 790 nm as compared to 810 nm in the absence of detergent, the latter being the wavelength used in the original procedure (1). The standard curve, Fig. 1, showed excellent linearity of at least up to 0.40/xmol orthophosphate, permitting the routine use of the concentration readout feature of the SP1800 spectrophotometer.