[10] Assay of inorganic phosphate, total phosphate and phosphatases

This chapter discusses the assay of inorganic phosphate, total phosphate, and phosphatases. The phosphomolybdate complex is reduced by ascorbic acid. The method is about seven times as sensitive as the Fiske–SubbaRow procedure and involves less pipetting. One can easily determine 0.01 micromole of phosphate. Pyrophosphate breaks down about 5% in the method and compounds such as glucose 1-phosphate also break down somewhat, so that the method is not very satisfactory for determining inorganic phosphate if labile phosphate esters are present in large excess. The sample of organic phosphate and a drop of magnesium nitrate solution in a small test tube are taken to dryness by shaking the tube in flame. The ashing procedure is rapid and is good for various biological materials and phosphate esters such as nucleic acid, carbohydrate phosphate esters, viruses, and phospholipids. The assay method of phosphatases for inorganic phosphate can be used as an assay for phosphatases hydrolyzing stable phosphate esters such as glucose-6-phosphate, ribose-5-phosphate, and histidinol phosphate. The enzyme incubation can be stopped with the one ascorbic-molybdate solution thus avoiding an extra pipetting.