Improving the stability of the malachite green method for the determination of phosphate using Pluronic F68

MARTÍNEZ GACHE, SANTIAGO A.; RECOULAT ANGELINI, ALVARO A.; SABECKIS, M. LURDES; GONZÁLEZ FLECHA, F. LUIS

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2020

0003-2697

The analytical determination of phosphateconstitutes a fundamental step for the evaluation of catalytic activities ofmany enzymes involved in the hydrolysis of phosphate-containing biomolecules.The most sensitive colorimetric methods to quantify phosphate are based onmeasuring the spectral changes produced by the adsorption of malachite green to12-molybdophosphoric acid. Malachite green methods are time-consuming becausethey require the preparation of color reagent on the day of use, due to its lowstability. In this work we propose a modification of the malachite green methodthat overcomes these problems and only requires a one-step, ready-to-use stocksolution including perchloric acid and Pluronic F68. The improved reactionmixture allowed the quantification of phosphate with a limit of detection of 0.22μM, a dynamical range up to 80 μMdepending on the optical path-length, and a molar absorptioncoefficient at 640 nm of 94000 M-1cm-1. Color developmentreaches a steady level within 30 min and remains constant for at least 2 h. Thehigh stability of the color reagent allows long-term storage for at least 1year. The optimized procedure is especially useful to measurephosphate-containing biomolecule levels and enzyme activities when low valuesare critical.