The determination of inorganic orthophosphate in biological systems |
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Authors: | A Atkinson AD Gatenby AG Lowe |
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Institution: | Department of Biological Chemistry, University of Manchester, Manchester, M13 9PL Great Britain |
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Abstract: | A simple and rapid method is described for determining Pi by spectrophotometric measurement of a soluble complex of phosphomolybdic acid and Cirrasol ALN-WF, a non-ionic detergent formerly known as Lubrol W. The measured complex has a molar extinction coefficient of 4.59 · 103 at 390 nm and little interference is found with relatively high concentrations of chelating agents, salts, and other compounds which interfere with most other Pi assays. Linearity is observed in the range 0–1.2 μmoles Pi and developed assay samples are stable for 8 h at 20 °C or 24 h at 4 °C. The method is suitable for use in the presence of moderate concentrations of protein or ATP.After suitable modification the assay can be used at pH 4.0. Sensitivity is reduced at this pH (εM, 390nm = 2.79 · 103) but linearity is maintained up to 1 μmole Pi and the coloured complex is stable for 4 h at 20 °C. The pH-4 procedure is suitable for measurement of Pi in the presence of very labile phosphate esters such as creatine phosphate.The phosphomolybdic acid-Cirrasol complex can be reduced at ambient temperature in both the above systems. A blue complex results with εM, 820nm of 9.9 · 103 at pH 4.0, and 1.8 · 104 under more acidic conditions. |
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