Polyphosphate Chain Length Determination in the Range of Two to Several Hundred P-Subunits with a New Enzyme Assay and 31P NMR.

Abstract

Currently, 31P NMR is the only analytical method that quantitatively determines the average chain length of long inorganic polyphosphate (>80 P-subunits). In this study, an enzyme assay is presented that determines the average chain length of polyphosphate in the range of two to several hundred P-subunits. In the enzyme assay, the average polyP chain length is calculated by dividing the total polyphosphate concentration by the concentration of the polyphosphate chains. The total polyphosphate is determined by enzymatic polyphosphate hydrolysis with Saccharomyces cerevisiae exopolyphosphatase 1 and S. cerevisiae inorganic pyrophosphatase 1, followed by colorimetric orthophosphate detection. Because the exopolyphosphatase leaves one pyrophosphate per polyphosphate chain, the polyphosphate chain concentration is assayed by coupling the enzymes exopolyphosphatase (polyP into pyrophosphate), ATP sulfurylase (pyrophosphate into ATP), hexokinase (ATP into glucose 6-phosphate), and glucose 6-phosphate dehydrogenase (glucose 6-phosphate into NADPH), followed by fluorometric NADPH detection. The ability of 31P NMR and the enzyme assay to size polyP was demonstrated with polyP lengths in the range from 2 to ca. 280 P-subunits (no polyP with a longer chain length was available). The small deviation between methods (-4 ± 4%) indicated that the new enzyme assay performed accurately. The limitations of 31P NMR (i.e., low throughput, high sample concentration, expensive instrument) are overcome by the enzyme assay that is presented here, which allows for high sample throughput and requires only a commonly available plate reader and micromole per liter concentrations of polyphosphate.

• Authors: Jonas Johannes Christ, Sabine Willbold, Lars Mathias Blank
• Journal: Analytical chemistry (Anal Chem) Vol. 91 Issue 12 Pg. 7654-7661 (06 18 2019) ISSN: 1520-6882 [Electronic] United States
• PMID: 31082217 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Diphosphates
• Polyphosphates
• Saccharomyces cerevisiae Proteins
• diphosphoric acid
• NADP
• Acid Anhydride Hydrolases
• Inorganic Pyrophosphatase
• exopolyphosphatase

Topics

• Acid Anhydride Hydrolases (metabolism)
• Diphosphates (analysis)
• Enzyme Assays (methods)
• Fluorometry
• Inorganic Pyrophosphatase (metabolism)
• Magnetic Resonance Spectroscopy (methods)
• NADP (analysis)
• Polyphosphates (analysis)
• Saccharomyces cerevisiae (enzymology)
• Saccharomyces cerevisiae Proteins (metabolism)