Improvement of biochemical methods of polyP quantification

Abstract

Polyphosphate (polyP) is an abundant and physiologically important biomolecule for virtually any living cell. Therefore, determination of changes in cellular content of polyP is crucial for its functional characterization. Determination of cellular polyP has been performed by many different methods, and the lack of a standardized procedure is possibly responsible for the large dispersion of results found in the relevant literature. For a relatively simple organism, such as the yeast Saccharomyces cerevisiae, this variation can be up to 12-fold. polyP extraction and determination of free phosphate released by enzymatic degradation of the polymer is a method quite common and relatively straightforward for polyP determination. By using the yeast S. cerevisiae as model, we have experimentally evaluated the different steps in this procedure in order to identify critical issues that might explain the disparate reported results. As the main output of this evaluation we propose a straightforward and robust procedure that can be used as gold standard protocol for cellular polyP purification and determination from unicellular organisms, thus providing consistency to measurements and facilitating inter-laboratory comparisons and biological interpretation of the results.

Keywords: Saccharomyces cerevisiae; neutral-phenol; polyphosphate; yeast.

Figure 1. FIGURE 1: High variability on polyP determination in S. cerevisiae.

(A) polyP content in mM concentration according to different authors. Values were converted to mM according to the following assumptions: i) The volume of the haploid cell is 42 fl, ii) One OD₆₆₀ unit equals to 2.6 x 10⁷ cells and represents 50 µg of RNA. The Mw for the [-PO₃^--] monomer is 79 g/mol. (B) Flow chart of this work, showing the different possibilities explored in each of the three parts needed for determining polyPs: extraction, purification and quantification.

Figure 2. FIGURE 2: polyP is unstable when strong acids are employed during the extraction process.

Relative amount of polyP after treatment with the different extraction solutions. Commercial polyP (4 μg) was incubated at different times with the different extraction solutions: (A) neutral-phenol at 4°C, (B) acid-phenol at 4°C, (C) 1 M H₂SO₄ at room temperature and (D) 1 M HClO₄ at 4°C. The mixes were neutralized, purified using affinity columns and the polyP eluted with MilliQ water. polyP amount was determined from the amount of Pi produced upon treatment with rPpx1. The graphs represent the percentage of polyP relative to time zero of each condition. Mean ± SEM from 3 independent experiments is shown.

Figure 3. FIGURE 3: Ethanol precipitation method yields a broader spectrum of polyP sizes than affinity column purification.

(A) PAGE and DAPI staining of polyP differently purified. polyP was extracted using the neutral phenol/chloroform procedure from a yeast pellet equivalent to 10⁷ logarithmically growing yeast cells. The aqueous phase was treated with DNAse and RNAse solution, and purified by affinity columns or by ethanol precipitation. The resulting polyP fractions: precipitated (in the case of the ethanol) and eluted and flow-through (in the case of the affinity column) were analyzed by PAGE followed by DAPI staining. (B) Percentage of polyP relative to the purification method. polyP amount was determined using the same fractions obtained in panel A. The graph represents the Mean ± SEM from 3 independent experiments. (C) Relative amount of polyP obtained after the precipitation of polyP in the presence of different monovalent salts, divalent salts and a carrier. Aqueous phase from panel A was used. The graph represents the percentage of polyP relative to precipitation with NaOAc. Mean ± SEM from 3 independent experiments is shown. GLC, glycogen.

Figure 4. FIGURE 4: rPpx1 activity is inhibited by the presence of DNA and RNA.

(A) Kinetic of the rPpx1 polyP digestion. rPpx1 (10 ng) was incubated with 250 ng of commercial polyP or yeast polyP, in 20 mM Tris-HCl pH 7.50 containing 5 mM magnesium acetate and 100 mM ammonium acetate at 37°C. Samples were taken at the indicated times to quantify the released Pi. Mean ± SEM from 3 independent experiments is shown. (B) rPpx1 activity on polyP in the presence of increasing amount of DNA. rPpx1 (10 ng) was incubated with 100 ng of commercial polyP in 20 mM Tris-HCl pH 7.50 containing 5 mM magnesium acetate and 100 mM ammonium acetate and at 37°C during 20 min with increasing concentrations of DNA (both circular and linear). The graph represents the released Pi in each condition. Mean ± SEM from 3 independent experiments is shown. (C) rPpx1 activity on polyP in the presence of increasing amount of RNA. Same experiment as in B, but with increasing concentrations of RNA. The graph represents the released Pi in each condition. Mean ± SEM from 3 independent experiments is shown. (D) Kinetics of rPpx1 polyP digestion in the presence of DNA and RNA. rPpx1 (1 ng) was incubated with 250 ng of yeast polyP previously treated or not with a DNAse/ RNAse solution in 20 mM Tris-HCl pH 7.50 containing 5 mM magnesium acetate and 100 mM ammonium acetate at 37°C. Samples were taken at the indicated times and the released Pi was quantified. Mean ± SEM from 3 independent experiments is shown.

Figure 5. FIGURE 5: Comparison of different polyP extraction and purification methods.

(A) Amount of polyP corresponding to 10⁷ logarithmically growing yeast cells extracted and purified with the following methods: neutral-phenol/chloroform and ethanol precipitation , acid-phenol/chloroform and ethanol precipitation , perchloric acid , and sulfuric acid and affinity columns . Mean ± SEM from 3 independent experiments is shown. (B) Scheme of the polyP extraction and purification protocol using neutral phenol and ethanol purification. In brackets appears the figure supporting this particular step. For details and tips see Materials and Methods.