A novel enzymatic quantification method for polyphosphate in phytoplankton

Abstract

Polyphosphate (polyP) is a basic biological polymer made up of linear chains of inorganic phosphate (Pi) units linked by high-energy phosphoanhydride bonds. The presence of polyP is ubiquitous, but its study has historically been ignored and limited by difficulties in extracting and quantifying polyP from cells. Recently published analytical polyP extraction and enzymatic quantification methods claim to be the “gold standard” for quantitative polyP research but have not yet been tested in species other than Saccharomyces cerevisiae. The adaptation of these methods for use in the quantification of polyP in phytoplankton was attempted. The molybdenum blue assay, using cloned and expressed exopolyphosphatase (EC 3.6.1.11) and pyrophosphatase (EC 3.6.1.1) enzymes, very accurately determined the concentration of polyP in a sample after its enzymatic hydrolysis to Pi. Analyte response is largely unaffected by buffer contamination with phenol or chloroform and sample dilution with Milli-Q water. The polyP extraction methods were unsuccessful and not able to replicate the results of the published protocol when attempted on polyP standards or Polysiphonia samples. An older polyP extraction protocol was used to successfully extract polyP from Synechocystis sp. PCC6803 but was unable to extract polyP standards. By combining these older extraction methods and newly published enzymatic quantification methods, the concentration of polyP stored within the cells of phytoplankton and cyanobacteria can accurately be measured for future studies, but the cause of the inability of either method to extract polyP standards is still unknown.