Biochemistry

Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the polyP values obtained.


Figure 1. Enzymatic hydrolysis of polyP

Inorganic polyphosphate (poly P) molecules, linear chains containing hundreds of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds are abundant in every cell in nature. These molecules are widely distributed among bacteria, including key pathogens, and eukaryotes, poly P is present in organelles, including nuclei, mitochondria, and vesicles.

Remarkable properties of this molecule as a polyanion have been discovered and have made it suited for a crucial role in the emergence of cells on earth. Poly P is essential for bacterial responses to stresses and starvation, motility, quorum sensing, biofilm formation, and virulence and essential for survival. Polymers of different lengths are present in different locations and have different roles in the cell.

A simple, rapid, and sensitive colorimetric microassay for inorganic phosphate (Pi) relies upon the absorption at 660 nm of a molybdenum blue complex that forms upon reduction of an ammonium molybdate-Pi complex in acid. The method for determination of total Pi uses plant tissues that have been ashed at 500 °C, whereas quantification of soluble Pi is performed with tissues extracted under mild acid conditions (which preserves acid-labile phosphate ester bonds).