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Polyphosphate chain length determination in the range of two to several hundred P-subunits with a new enzyme assay and P NMR 31

Jonas Johannes Christ, Sabine Willbold, and Lars M. Blank Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00567 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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Analytical Chemistry

Polyphosphate chain length determination in the range of two to several hundred P-subunits with a new enzyme assay and 31P NMR Jonas Johannes Christ a, Sabine Willbold b, and Lars Mathias Blank a, * a Institute

of Applied Microbiology – iAMB, Aachen Biology and Biotechnology – ABBt, Worringer Weg 1, RWTH Aachen University, D-52074, Aachen, Germany Central Institute for Engineering, Electronics and Analytics, Analytics (ZEA-3), Wilhelm-Johnen-Straße, D-52428 Jülich, Germany b

To whom correspondence should be addressed: Prof. Dr. Lars Mathias Blank, Institute of Applied Microbiology – iAMB, Aachen Biology and Biotechnology – ABBt, RWTH Aachen University, Worringer Weg 1, D-52074, Aachen, Germany, Telephone: 0049 241 80 26600, FAX: 0049 241 80 622180, E-mail: [email protected] *

Keywords: inorganic polyphosphate, chain length, orthophosphate, colorimetric assay, fluorometric assay, enzymatic assay

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 6phosphate 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 of 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.

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Introduction Inorganic polyphosphate (polyPn, where n is the average chain length) is the linear polymer of orthophosphate (Pi) with a chain length of at least 2 and up to 1000 P-subunits, and is found in nearly all living organisms (1-3; an exception is, for example, Enterococcus faecalis 1). Cyclic polyP, with a chain length of 3 to 12 P-subunits, can be synthesized chemically and is formed spontaneously during hydrolysis of long-chain linear polyP 2. In comparison with linear polyP, cyclic polyP has no hydroxyl group with neutral pKa value at the end groups, and does not possess the (biotechnologically valuable) higher valent cation-complexing ability. In biological systems, no function of cyclic polyP has been found yet 2. Because the physicochemical properties of polyP depend on its chain length 4, 5, polyPs of varying chain lengths have specific functions in vivo (6, p. 125ff in Ref. 2). Furthermore, different industrial applications of polyP require defined average polyP chain lengths 4. Thus, both the total polyP concentration and the average polyP chain length have garnered interest in studies on polyP 7. It should be noted that other important properties of polyP, such as the chain length distribution and counter-ion composition, are not covered in this study. An enzymatic method for specific total polyP determination is available (abbreviated as the PPX/IPP assay 8). For polyP chain length determination, seven methods exist: end group titration, ion chromatography, capillary electrophoresis, the PPX/IPP assay, polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography, and 31P nuclear magnetic resonance (31P NMR). End group titration is limited to pure polyP and gram amounts thereof, rendering this method unsuitable for the analysis of biological samples 9. Ion chromatography and capillary electrophoresis are restricted to polyP with a maximum chain length of 30 and 45 P-subunits, respectively 9-12. The PPX/IPP assay is based on the differential hydrolysis of a polyP sample with the enzyme Saccharomyces cerevisiae exopolyphosphatase 1 (scPpx1p; EC 3.6.1.11; reaction 1 in Figure 1), and a mixture of scPpx1p and S. cerevisiae inorganic pyrophosphatase 1 (scIpp1p; EC 3.6.1.1; reaction 2 in Figure 1). Subsequently, the released Pi is determined colorimetrically. The PPX/IPP assay, however, is limited to the determination of a polyP length in the range of 2 to 80 P-subunits 8. In PAGE, the polyP is separated on a polyacrylamide gel by an electric current and is subsequently stained 13. PAGE provides information on the chain length distribution. However, PAGE requires a sequencing apparatus, is time-consuming, allows for the analysis of only ca. 20 samples at a time, and gives only non-quantitative results. With calibrated standards, the average polyP chain length can be estimated with PAGE 13, 14. Similar to PAGE, size exclusion chromatography provides information on the chain length distribution, and is dependent on polyP size standards 15-18. In 31P NMR, polyP gives three resonance peaks: terminal P (PP1) at ca. -7 ppm, P at position two (PP2) and three (PP3) in the polyP chain at ca. -20.17 to -21.7 ppm, and P at position four or higher in the polyP chain (PP4) at ca. -22.5 ppm (19, p. 27 in Ref. 2). 31P NMR differentiates between linear polyP, cyclic polyP, and Pi 20, 21. Lindner et al. proposed a formula to calculate the average polyP chain length from the peak areas of PP1 to PP4 (Eq. 1 in Figure 2; 20). To overcome the analytical challenge of peak overlap, the formula uses PP1, PP2, or PP3 in the denominator, depending on which has the best resolution. With NMR, the average polyP chain length is quantitatively determined. However, NMR requires a special instrument, is limited to the analysis of one sample at a time, and is completed in several minutes to an hour per sample. Sometimes, PP1 cannot be resolved due to the overlap of β-P and γ-P with nucleotide diphosphate and nucleotide triphosphate, respectively. Further, 31P NMR lacks sensitivity for determinations down to micromoles per liter 19. Nevertheless, the ability of 31P NMR to quantitatively size polyP up to hundreds of P-subunits has been, until now, unrivalled. In conclusion, there is a need for an assay that is quantitative, allows for high throughput processing, can quantify in the range of micromoles per liter, is insensitive to biological impurities, and requires only commonly available laboratory equipment. The average polyP chain length can be calculated by dividing the total polyP concentration by the concentration of the polyP chains (Eq. 2 in Figure 2). These two variables were quantified in this study by two enzymatic assays (Figure 1). Total polyP was quantified according to a recent study (Figure 1A; 8). Every polyP was hydrolyzed irreversibly by scPpx1p into one polyP2 and (n - 2) Pi (reaction 1 in page 2 of 19


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Figure 1). The molecular reasons for the inability of scPpx1p to cleave polyP2 are discussed in Ref. 22 (see also Ref. 23 for a biochemical characterization of scPpx1p). At the same time, polyP2 was irreversibly hydrolyzed by scIpp1p into two Pi (reaction 2 in Figure 1). The hydrolysis of a polyP sample with scPpx1p and scIpp1p, and the subsequent colorimetric Pi detection (reaction 3 in Figure 1) allowed for the quantification of the total polyP concentration. Overall, polyPn was quantified as n blue complexes (reaction 4 in Figure 1). The polyP chain concentration was detected fluorometrically (Figure 1B). From every polyP chain, scPpx1p produced one polyP2 (reaction 5 in Figure 1). PolyP2 reacted with adenosine 5’phosphosulfate to produce ATP and sulfate (catalyzed by ATP sulfurylase; EC 2.7.7.4; reaction 6 in Figure 1). Although the reaction of ATP sulfurylase is reversible, in vitro the enzyme favors the reaction toward ATP (equilibrium constant 10-8, see Ref. 24). Hexokinase (EC 2.7.1.1) irreversibly transferred one Pi from ATP to glucose (reaction 7 in Figure 1). Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) reversibly produced one fluorescent NADPH per glucose 6-phosphate from one non-fluorescent NADP+ (reaction 8 in Figure 1; 25). The NADPH concentration was quantified based on its fluorescence. The overall reaction is shown in reaction 9 in Figure 1. The quantification of polyP2 by coupling ATP sulfurylase, hexokinase and glucose 6-phosphate dehydrogenase was inspired by Drake et al. 26. The first goal of this study was to develop an enzyme assay that allows for the determination of the average polyP chain length in the range of two to several hundred P-subunits. In the second aim, both the performance of the enzyme assay and 31P NMR for the determination of the polyP chain length was evaluated with polyPs that comprised 2 to ca. 280 P-subunits.

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Materials and methods Chemicals and enzymes PolyP3 (penta-sodium triphosphate, item number ACRO393961000) was obtained from VWR (Radnor, PA, USA). PolyPs “Budit 7” (average chain length: 13; 8) and ”Budit 4” were a kind gift from Budenheim (Budenheim, Germany). polyP2 (tetra-sodium diphosphate decahydrate, item number T883), black polystyrene 96-well microtiter plates (item number CEK8), and transparent polystyrene 96-well microtiter plates (item number 9293), NADP+ (disodium salt, ≥ 97 % purity, item number AE13), salmon sperm DNA, and RNA sodium salt were obtained from Carl Roth (Karlsruhe, Germany). PolyPs “p100”, “mode 155”, “mode 385”, and “p700” were a kind gift from Dr. James H. Morrissey (University of Michigan Medical School). The polyPs with a homogenous chain length were polyP2 and polyP3. All other polyPs were a mixture of varying chain lengths. The polyP concentrations (including polyP2) are reported as Pi. Adenosine 5’-phosphosulfate (sodium salt, 95 % purity, item number 21226) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Sterile filtration was performed with 0.2-µm-pore polyethersulfone membranes (Nalgene, Thermo Fisher Scientific, Waltham, MA, USA). ATP-free water was prepared by sterile-filtering and autoclaving bidistilled water. ATP sulfurylase (from S. cerevisiae, item number M0394L) was purchased from NEB (Ipswich, MA, USA). Hexokinase (from S. cerevisiae, item number H6380) and glucose 6-phosphate dehydrogenase (from Leuconostoc mesenteroides, item number G8529) were obtained from SigmaAldrich. scPpx1p and scIpp1p were prepared according to Ref. 8 (NCBI accession numbers L28711 and NP_009565, respectively). scPpx1p, scIpp1p, and glucose 6-phosphate dehydrogenase were dissolved and / or diluted in filter-sterilized 0.1 % (w/v) bovine serum albumin (protease free), 50 % (v/v) glycerol, 20 mM Tris-Cl, and 50 mM KCl at pH 7.5. ATP sulfurylase was diluted in filter-sterilized 10 mM Tris-Cl, 50 mM NaCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 50 % (v/v) glycerol at pH 7.5. All enzymes were stored at -20°C. The specific activities of scIpp1p and scPpx1p were assayed by measuring the Pi that was released from polyP2 and Budit 7, respectively, during a 10 min period. The final assay concentrations were as follows: 5 mM magnesium acetate, 20 mM Tris-Cl, 50 mM ammonium acetate, 1 mM polyP2 or Budit 7, and pH 7.5. The released Pi was quantified colorimetrically, as described below. One unit of scIpp1p was the amount of enzyme that generated 1 μmol of Pi per minute at 37°C. One unit of scPpx1p activity was defined as the release of 1 pmol Pi per minute at 37°C 23. The activities of the other enzymes were taken as stated by the manufacturer. Enzymatic determination of the total polyP concentration Total polyP was determined by enzymatic hydrolysis with scPpx1p and scIpp1p, and subsequent colorimetric Pi detection according to Ref. 8. Briefly, the samples for the total polyP analysis were diluted in dilution buffer (filter-sterilized 1 mM sodium 3-(N-morpholino)propanesulfonic acid (MOPS) and 0.1 mM EDTA at pH 7, prepared with ATP-free water). A total of 50 µl enzyme reaction buffer (15 mM magnesium acetate, 60 mM Tris-Cl, 150 mM ammonium acetate, pH 7.5, filter-sterilized), that contained 11,000 U scPpx1p and 0.013 U scIpp1p, was added to 100 µl sample with a concentration of 5 to 200 µM polyP (optimal: 100 µM polyP) in a transparent microtiter plate. After an incubation at 1 h ± 5 min at 37°C, 50 µl Pi detection reagent (2.4 mM ammonium heptamolybdate, 600 mM H2SO4, 0.6 mM antimony potassium tartrate, and freshly added ascorbic acid up to 88 mM) was added. Then, after an incubation for exactly 2 min at room temperature, the absorbance was read at 882 nm, which indicated the Pi concentration. The KH2PO4 standards (20, 65, 110, 155, and 200 µM Pi in dilution buffer) were treated as samples, except that no blank was measured. For every sample, a blank without scPpx1p and scIpp1p was tested. The blank was equivalent to the Pi concentration of the sample. The Pi concentrations were calculated with a calibration curve. The total polyP minus the blank concentration was calculated per Eq. 3 in Figure 2. page 4 of 19


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Enzymatic determination of the polyP chain concentration Both sodium MOPS buffer (50 mM, pH 7) and TMG buffer (450 mM Tris-acetate, 30 mM magnesium acetate, 12 mM glucose, pH 7.6) were prepared with ATP-free water and filter-sterilized. The aliquots of adenosine 5’-phosphosulfate (2 mM in MOPS buffer), NADP+ (20 mM in MOPS-buffer), and 100 µM polyP2 were stored at -20°C. The polyP2 detection reagent was freshly prepared by mixing the following per reaction: 25 µl TMG buffer, 5 µl adenosine 5’-phosphosulfate (2 mM), 5 µl NADP+ (20 mM), 1 µl scPpx1p (33,000 U * µl-1), 1 µl ATP sulfurylase (0.075 U * µl-1), 1 µl hexokinase (0.075 U * µl-1), 1 µl glucose 6-phosphate dehydrogenase (0.075 U * µl-1), and 11 µl ATP-free water. The samples and polyP2 standards were dissolved and diluted in dilution buffer. A total of 100 µl of sample, which contained 1 to 10 µM polyP2 (optimal: 5 µM), was pipetted into a black microtiter plate. After the addition of 50 µl polyP2 detection reagent, the plate was incubated for 55 ± 5 min at room temperature and then for 5 min at 25°C in the plate reader, to equilibrate the temperature. Next, the fluorescence (excitation at 340 nm with a bandwidth of 9 nm, emission at 460 nm with a bandwidth of 9 nm, automatic gain adjustment scaled to the high wells “10 µM polyP2” with maximum scale value) was read, which reflected the NADPH concentration. For each sample, a blank without scPpx1p and ATP sulfurylase was measured. Every day, the concentration of the 100 µM polyP2 standard was verified by determining the total polyP. The polyP2 standards (1, 2.5, 5, 7.5, 10 µM polyP2 in dilution buffer) were prepared daily from the 100 µM polyP2 standard, and treated as samples, except that no blank was measured. The fluorescence values were converted to polyP2 concentrations using a calibration curve. The polyP chain concentration was calculated according to Eq. 4 in Figure 2. As for every polyP in this study, the concentration of polyP2 is reported as the monomer (i. e., µM Pi). However, the chain concentration is equal to the polyP2 polymer concentration (i. e., µM polyP2). To convert between the monomer and the polymer concentrations, a factor of 0.5 was included in Eq. 4 in Figure 2. The “blank” in Eq. 4 (Figure 2) refers to a measurement without ATP sulfurylase and scPpx1p, which allows contaminating NADPH, ATP and glucose 6phosphate to be subtracted. Enzymatic determination of the polyP concentration, the average polyP chain length, and the Pi concentration The workflow of the enzyme assay that has been developed here is shown in Figure 3. The test was divided into the determination of the polyP chain concentration (Figure 3A and B), and the determination of the total polyP concentration (Figure 3C and D). Figure 3D describes the measurement of the blank of Figure 3C and the Pi concentration. Before this assay was performed, a pre-test was performed in single measurements, to determine the appropriate dilutions. The sample concentration that was required for Figure 3A was estimated, if the approximate chain length was known (Eq. 5 in Figure 2). A more wasteful, but time-saving, alternative was to omit the pre-test, and directly run the main test with several sample dilutions. In the main test, blanks and samples were analyzed in triplicate for the total polyP assay and chain concentration assay. The KH2PO4 and polyP2 standards were analyzed in duplicate. The 100 µM polyP2 standard was analyzed in six measurements in the total polyP assay, because this was the reference for the calibration of the chain concentration assay. The appropriate dilutions for Figure 3A and B were not the same, because the latter was the blank of the former and because the linear range of polyP2 detection was narrow (i. e., 1 log). For the total polyP and its blank determination, 100 µM total polyP, which was equal to the middle of the linear range of the Pi detection, was chosen (Figure 3C and D). For the detection of Pi in the presence of polyP, the total polyP concentration should not exceed 200 µM, to avoid non-enzymatic polyP hydrolysis during Pi detection. The average polyP chain length was calculated per Eq. 2 in Figure 2. The concentrations in this equation refer to the undiluted sample.

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31P

NMR for the determination of the average polyP chain length

The solution 31P NMR spectra were obtained using a Bruker 600-MHz spectrometer that was operated at a 31P frequency of 242.95 MHz, and equipped with a prodigy-probe (a broadband CryoProbe that uses Ncooled RF coils and preamplifiers to deliver sensitivity enhancement over room temperature probes by a factor of 2 to 3 for nuclei from 15N to 31P). The samples were measured with a D2O-field lock at room temperature, and the chemical shifts were referenced to 85 % orthophosphoric acid (0 ppm). The NMR parameters that were generally used were as follows: 32 K data points, 60 s repetition delay, 0.7 s acquisition time, 30° pulse width and 64 scans. The peak areas were calculated by integration of the spectra that were processed with the 1 Hz line-broadening, using Bruker Topspin software and in cases, in which the peak areas could not be integrated unambiguously, MestReNova was used for curve fitting. The individual P compounds were identified based on their chemical shifts and splits. These compounds included Pi (1-2 ppm), polyP2 (-7 to -8 ppm), polyP (-6 to -8, -20 to -23 ppm) and cyclic polyP from approximately -23 to -25 ppm. The accurate values of the chemical shifts of these signals depended on pH and the residual concentration of the divalent cations in the solution. The chain length of the linear polyP was calculated from the signal intensities of PP1, PP2, PP3, and PP4. In cases, in which the integration of the signal of the core phosphate group (PP4) was not possible due to overlays, the signals were fitted. This step also applied to the signal for the polyP2. The average polyP chain length for polyPn ≥ 3 was calculated according to Eq. 1 in Figure 2. The overall average polyP chain length was calculated as the weighted mean of polyPn ≥ 3 and polyP2 (Eq. 6 in Figure 2). The relative error of the method was ca. 2 %.

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Results The total polyP and Pi determinations (Figure 3C and D; Figure 1A) were already established in a previous study 8. The present study developed a method to measure the polyP chain concentration (Figure 3A and B; Figure 1B). The combination of the total polyP and polyP chain determinations allowed the quantification of the average polyP chain length. Because the determination of the polyP chain concentration was an endpoint assay, it was possible to use enzymes and substrates in excess. With regard to the upper limit of detection (10 µM polyP2), excesses of 10-fold for adenosine 5’-phosphosulfate, 300fold for glucose, and 100-fold for NADP+ were chosen, to drive the reactions to completion. ATP sulfurylase, hexokinase, and glucose 6-phosphate dehydrogenase were used at a 4500-fold excess, to accommodate for the variance in enzyme activity, and to counteract inhibiting substances. Further, the rapid polyP2 consumption by these enzymes pulled the reaction of scPpx1p to completion. The reaction speed, starting from the intermediate products (i. e., polyP2, ATP, and glucose 6-phosphate; reactions 6 to 8 in Figure 1), was sufficiently fast that the reaction was completed in under a minute (data now shown). In the chain concentration assay, one polyP2 was expected to be released per polyP chain. Thus, the required polyP concentration for the assay depended on the polyP chain length (Eq. 5 in Figure 2; reaction 5 in Figure 1). For example, polyP2 was used at 5 µM, whereas polyP380 was used at 950 µM, to elicit the same polyP2 concentration (5 µM) after treatment with the enzymes. Thus, for scPpx1p the highest substrate concentration was given for the longest polyP. Seven thousand U scPpx1p per reaction sufficed to hydrolyze 1 mM of the longest available polyP (polyP mode 384) to polyP2 in 1 h. To accommodate for the variance in enzyme activity and even longer polyP, a 4.7-fold excess (33,000 U scPpx1p * reaction-1, which was the highest available enzyme concentration) and one hour incubation time were chosen for the standard assay. The authors previously showed that 2,200 U scPpx1p per reaction suffice to hydrolyze 200 µM polyP3 under similar reaction conditions 8. Full conversion of polyP3 (one of the shortest polyPs) by scPpx1p was, thus, ensured. The fluorescence intensity of pure NADPH in water decreased with rising temperature. This effect was notable when measuring the NADPH fluorescence over time in a microtiter plate, which was at room temperature and then placed in a 37°C plate reader (data not shown). To keep the microtiter plate at a constant temperature, the assay was performed at room temperature and the microtiter plate was preincubated for 5 min at 25°C in the plate reader prior to the fluorescence being read. A linear relationship between the polyP2 concentration and the fluorescence was observed in the range of 1 to 10 µM polyP2. The signal to blank ratio was 1.7 for 1 µM polyP2 and 8.0 for 10 µM polyP2. The regression coefficient R² was excellent (0.999). The limit of quantification (10 * standard error of regression * (slope)-1) was 1.0 µM polyP2. Two hypotheses were confirmed, to ensure proper function of the chain concentration assay. The first hypothesis was as follows: under standard assay conditions, ATP sulfurylase catalyzes ATP formation only from polyP2, but not from polyPn ≥ 3. If ATP sulfurylase also acts on polyPn ≥ 3, ATP would form from a polyP, which does not contain polyP2. This hypothesis was tested with polyP3, because, according to the manufacturer, it contained only polyP with a chain length of 3 P-subunits. Under standard assay conditions, but without scPpx1p, 3.7 µM polyP2 was detected from 100 µM polyP3. This small amount of polyP2 was explained by the non-enzymatic hydrolysis of polyP3 to polyP2 or by the contamination of the polyP3 with polyP2. The latter speculation was later verified by 31P NMR (Table 2). Thus, hypothesis one was confirmed. The second hypothesis was as follows: under standard assay conditions, scPpx1p hydrolyzes polyPn ≥ 3, but not polyP2, indicating that no polyP2 is hydrolyzed by scPpx1p in the relevant polyP2 range (1 to 10 µM polyP2). To test this hypothesis, the polyP2 standards were analyzed for their polyP2 concentration at various scPpx1p concentrations (0; 2,200; 7,000; 33,250; 66,500; and 133,000 U scPpx1p * reaction-1). The hydrolysis of polyP2 through scPpx1p would have been visible in a decreased slope of the polyP2 concentration-fluorescence-calibration curve in comparison with a calibration curve, from which scPpx1p was omitted. The slope of the calibration curve fell by -0.028 % per 1,000 U PPX per reaction. This indicated that the slope was essentially the same for all scPpx1p concentrations. Thus, hypothesis two was confirmed. At this point, the assay development was complete. page 7 of 19



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The interference of certain substances on the enzyme assay was investigated (Table 1). An interference of ≤ 5 % was considered to be acceptable. NaCl was tested, to demonstrate that sea water is a possible sample matrix. The salt concentration of sea water is ca. 600 mM NaCl. One hundred and 400 mM NaCl had no effect on the enzyme assay. At 750 mM NaCl the chain concentration assay was inhibited by -10 %, which resulted in a +13 % increase in the measured chain length. The enzyme assay requires micromole per liter polyP concentrations (e. g., 150 µM polyP for polyP54). Thus, polyP samples in sea water probably must be diluted, effectively reducing the salt concentration. Concluding, analysis of polyP in sea water should be possible. Sulfate and ADP were tested as potential inhibitors of the enzymatic reactions that are depicted in reaction six and seven in Figure 1, respectively. Sulfate concentrations at ten-fold higher and lower concentrations than the effective sample concentration of adenosine 5’phosphosulfate (100 µM) were tested, and had no effect on the enzyme assay. ADP concentrations at 1-, 0.1-, and 0.01-fold the effective sample concentration of glucose (3,000 µM) were tested. Thirty µM ADP showed no interference. As measured in the blank of the chain concentration assay, ADP was contaminated with 2.1 molar percent ATP, preventing analysis of the interfering properties of 300 and 3,000 µM ADP. In theory, the influence of ADP should be minimal, because the hexokinase reaction is irreversible. DNA and RNA were tested as interfering substances, because they are often co-purified with polyP from biological samples. Twenty and 200 µM DNA and RNA had no effect. Two thousand µM DNA or RNA interfered strongly with the enzyme assay. For the analysis of biological samples that contain high ratios of nucleic acid to polyP (i. e., > 4 g nucleic acid to 1 g polyP), this effect should be considered (in this case, the use of RNase and DNase during polyP extraction is recommended). It was uncertain why there was a measurable chain concentration blank for DNA (0.4 molar percent NADPH equivalent with regard to the DNA monomer). None of the chain concentration blank-quantifiable substances (i. e., ATP, glucose 6-phosphate and NADPH) were expected from the salmon sperm DNA. In summary, the enzyme assay was sufficiently robust for the analysis of most biological samples. Seven polyP standards were analyzed for their mean polyP chain length with the enzyme assay (column 2 in Table 2). For the enzyme assay, coefficients of variance (CV; standard deviation divided by the mean) ± standard error of the CV were 2.1 ± 0.3 % for inter-assay, 3.1 ± 0.5 % for intra-chain concentration assay, and 1.5 ± 0.2 % for intra-total polyP assay. The low coefficients of variance indicated that the enzyme assay was reproducible. No significant correlation between the inter-assay CV and the chain length was found (slope: 0.003 CV [%] per 1 P-subunit, multiple correlation coefficient r = 0.289, p = 0.529). Concluding, the enzyme assay showed reproducible performance for short polyP as well as for very long polyP. The chain length of the same seven polyPs was also quantified by 31P NMR (Table 2). A highly significant positive correlation between the chain lengths that were determined by both methods was noted (slope: 1.0897 P-subunits determined with 31P NMR per P-subunit determined with the enzyme assay, r = 0.998, p = 1.3*10-7), confirming that these methods generated similar results. The deviation between methods was -4 ± 4 % (mean ± standard error of the CV). No significant correlation between the deviation between methods and the polyP chain length was observed (slope: -0.067 deviation between methods [%] per P-subunit, r = 0.64, p = 0.119). 31P NMR was used to quantify the content of cyclic polyP relative to the linear polyP (column 5 in Table 2). A highly significant negative correlation between the content of cyclic polyP and the deviation (negative deviation = chain length31P NMR < chain lengthenzyme assay) between methods was found (slope: -3.1 % deviation between both methods per 1-fold excess of cyclic to linear polyP, r = 0.914, p = 0.003). In other words, a high cyclic polyP content gave a lower reading for the chain length by 31P NMR compared with the enzyme assay.

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337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388


Discussion Advantages and disadvantages of the enzyme assay in comparison with 31P NMR For the first time, the chain length of polyP in the range of 2 to ca. 280 P-subunits was determined enzymatically. The determined chain lengths were consistent between 31P NMR and the enzyme assay. The enzyme assay measured somewhat longer chain lengths in the presence of cyclic polyP in comparison with the values that were obtained with 31P NMR. This result was unexpected, because scPpx1p does not hydrolyze cyclic polyP 8, 23. End group titration is a method for sizing highly pure polyP. In this approach, the total polyP concentration is determined by acidic hydrolysis and subsequent colorimetric Pi detection. The polyP chain concentration is measured by acid-base titration of the neutral hydroxyl groups at the polyP end groups of linear polyP 8. Previously, the chain length of the same batch of Budit 4, as used in the present study, was determined to comprise 21 P-subunits by end group titration 8. This finding agrees with the enzyme assay, but not with 31P NMR (Table 2). It remained unclear which method was influenced by cyclic polyP. Further studies should test different concentrations of cyclic polyP as an interfering substance. Even at a very high cyclic polyP content (e. g., 6.35-fold more cyclic polyP than linear polyP for polyP mode 100), the deviation between the enzyme assay and 31P NMR peaked at -19 %, which was tolerable (Table 2). The polyPs in the presented study were prepared by chemical synthesis, which is prone to producing cyclic polyP (see Ref. 1, pp. 6-7 in Ref. 2, Table 10-1 in Ref. 27, and Figure 40.14 in Ref. 28). The amount of cyclic polyP in biological polyP should be investigated in future studies. In summary, 31P NMR and the enzyme assay can be used to size polyP. 31P NMR has the advantage of quantifying cyclic polyP. The enzyme assay cannot detect cyclic polyP. The sample throughput of 31P NMR is low (min to h per sample), the required sample concentration is high (mM range), and an expensive instrument is required. The enzyme assay allows higher throughput (hundreds of samples per day), requires only a low sample concentration (µM range) and common laboratory equipment (multi-channel pipette, plate reader, 37°C incubator). Advantages and disadvantages of the enzyme assay in comparison with the PPX/IPP assay The recently published PPX/IPP assay sizes polyP in the range of 2 to 80 P-subunits 8. However, with greater chain lengths, the PPX/IPP assay becomes increasingly inaccurate, because the difference in Pi concentration between the scPpx1p and scPpx1p/scIpp1p wells becomes hard to resolve analytically. In the PPX/IPP assay, a significant increase in inter-assay CV was noted (an increase of 0.67 CV [%] per Psubunit, r = 0.964, p = 0.0018; 8). In other words, for polyP3 a CV of 0.5 % and for polyP28 a CV of 17 % was measured 8. In the enzyme assay from this study, the CV remained unchanged at ca. 2 % over the entire range of polyP lengths (increase of 0.003 CV [%] per P-subunit), which is a major advantage over the PPX/IPP assay. The enzyme assay from the presented study is based on two independent assays (i. e., total polyP and chain concentration assay). The PPX/IPP assay, in contrast, is “one” assay, which makes it easier to perform and less prone to inter-assay calibration errors. Further, the PPX/IPP assay does not coquantify ATP, glucose 6-phosphate and NADPH. In conclusion, for short polyP (2 to ca. 30 P-subunits), the PPX/IPP assay is a viable alternative to the enzyme assay from this study. PolyP chain length standards for PAGE The polyPs “p100”, “mode 155”, “mode 385”, and “p700” are used as chain length standards for PAGE (see, for example, Ref. 6, 14 for the application of similar polyP standards in PAGE). The numbers in the name indicate the average size that was determined with PAGE. The average polyP chain lengths of these standards were determined in this study to be 44, 5, 297, and 50 P-subunits by 31P NMR, respectively. Certain polyacrylamide gel concentrations allow for the resolution of only a certain range of polyP lengths (the longer the polyP, the lower the required polyacrylamide concentration). The separation of, for page 9 of 19



389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430

example, a polyP with an average chain length of 500 P-subunits on a low-strength (2 %) polyacrylamide gel would not show contaminating polyP with a chain length of < 300 P-subunits 13. The polyPs that are mentioned above were prepared by fractional precipitation of a heterogeneous polyP mixture 29. During the preparation, the contaminating short chain polyP was probably not removed entirely. This short polyP is not visible on the low strength polyacrylamide gels that are required for longer chain polyP, but is detected by 31P NMR and the enzyme assay. These findings demonstrate the difficulty of producing polyP length standards for PAGE and validating their length. ATP detection with luciferase, or hexokinase and glucose 6-phosphate dehydrogenase In the polyP chain concentration assay, ATP, that is produced by ATP sulfurylase, could have been quantified using firefly luciferase by reading the luminescence (Eq. 7 in Figure 2; 30). This reaction uses only one enzyme in comparison with the present study, which used hexokinase and glucose 6-phosphate dehydrogenase. There are two advantages of the luciferase detection system: Firstly, it has a wider linear range (2.5 logs, 1 nM to 500 nM; 30) versus the present system (1 log, 1 to 10 µM). Fewer dilutions have to be tested, to be within the linear range. In addition, because the linear range is more dilute, fewer chemicals and enzymes need to be used. Secondly, the luciferase system does not co-quantify glucose 6phosphate and NADPH. Conversely, there are three disadvantages of the luciferase system: Firstly, it is a kinetic system. Light is constantly produced by slowing down the reaction speed of the luciferase. This is achieved by adding the competitive inhibitor L-luciferin, reducing the oxygen concentration through reducing agents (e. g., dithiothreitol), using a low luciferase concentration, or by driving the reaction in the reverse direction through the addition of polyP2 31. Setting up a luciferase system, to emit a constant amount of light, is a delicate task, which can be disrupted easily by inhibiting substances 31. The here presented assay was developed for robustness. It is an endpoint assay that uses tremendous excesses of substrates and enzymes. Secondly, the here used system requires the addition of only one reagent. The luciferase system needs two (namely, ATP sulfurylase, an incubation step for scPpx1p to hydrolyze polyP, and luciferase). Thirdly, by itself luciferase produces polyP2 at a low rate. Because the task of the presented assay was to detect polyP2, which is released by scPpx1p, a positive feedback loop would form. Possible applications for the enzyme assay Endopolyphosphatases (EC 3.6.1.10) cleave polyP chains at non-terminal phosphoanhydride bonds, increasing the chain concentration at a constant total polyP. Kowalczyk and Phillips published a method for determining endopolyphosphatase activity that combines an enzyme assay and PAGE 32. The chain concentration assay from this study is a simpler alternative. The combination of analytical polyP extraction from living cells and the here presented enzyme assay allows for comprehensive Pi and polyP quantification as well as polyP length determination (see Ref. 33 for an analytical polyP extraction from S. cerevisiae). A liquid handling station with a plate reader, which reads absorbance and fluorescence, would allow for full automation of the presented enzyme assay. Concluding, the here presented enzyme assay can be used in all fields of polyP research that aim to determine the polyP chain length.

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431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452


Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contribution SW performed the 31P NMR experiments. JJC conducted all of the other experiments. JJC and LMB wrote the manuscript. LMB initiated and coordinated the study. All authors read and approved the final manuscript. Funding This work was supported by the Deutsche Bundesstiftung Umwelt (DBU). Acknowledgments We would like to thank Dr. Rainer Schnee and the Chemische Fabrik Budenheim KG for supplying us with polyP standards, and providing support regarding the analytical and conceptual questions. Further, the authors would like to thank Dr. James H. Morrissey (University of Michigan) for the polyP standards and his helpful advice. The authors would like to thank the anonymous reviewers for their helpful and constructive comments.

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Literature 1. Harold, F. M., Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriol. Rev. 1966, 30 (4), 772-794. 2. Kulaev, I. S.; Vagabov, V. M.; Kulakovskaya, T. V., The biochemistry of inorganic polyphosphates. 2nd ed.; John Wiley & Sons, Ltd: West Sussex, England, 2005. 3. Rao, N. N.; Gomez-Garcia, M. R.; Kornberg, A., Inorganic polyphosphate: essential for growth and survival. Annu. Rev. Biochem. 2009, 78, 605-47. 4. Christ, J. J.; Blank, L. M., Saccharomyces cerevisiae containing 28% polyphosphate and production of a polyphosphate-rich yeast extract thereof. FEMS yeast research 2019, 19 (3). 5. Kulakovskaya, T. V.; Vagabov, V. M.; Kulaev, I. S., Inorganic polyphosphate in industry, agriculture and medicine: Modern state and outlook. Process Biochemistry 2012, 47 (1), 1-10. 6. Smith, S. A.; Choi, S. H.; Davis-Harrison, R.; Huyck, J.; Boettcher, J.; Rienstra, C. M.; Morrissey, J. H., Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood 2010, 116 (20), 4353-9. 7. Majed, N.; Li, Y.; Gu, A. Z., Advances in techniques for phosphorus analysis in biological sources. Curr. Opin. Biotechnol. 2012, 23 (6), 852-9. 8. Christ, J. J.; Blank, L. M., Enzymatic quantification and length determination of polyphosphate down to a chain length of two. Anal. Biochem. 2018, 548, 82-90. 9. Baluyot, E. S.; G. Hartford, C., Comparison of polyphosphate analysis by ion chromatography and by modified end-group titration. J. Chromatogr. A 1996, 739 (1), 217-222. 10. Ohtomo, R.; Sekiguchi, Y.; Kojima, T.; Saito, M., Different chain length specificity among three polyphosphate quantification methods. Anal. Biochem. 2008, 383 (2), 210-6. 11. Stover, F. S., Capillary electrophoresis of longer-chain polyphosphates. J. Chromatogr. A 1997, 769 (2), 349-351. 12. Wang, T.; Li, S. F. Y., Separation of synthetic inorganic polymers of condensed phosphates by capillary gel electrophoresis with indirect photometric detection. J. Chromatogr. A 1998, 802 (1), 159165. 13. Clark, J. E.; Wood, H. G., Preparation of standards and determination of sizes of long-chain polyphosphates by gel electrophoresis. Anal. Biochem. 1987, 161 (2), 280-90. 14. Smith, S. A.; Wang, Y.; Morrissey, J. H., DNA ladders can be used to size polyphosphate resolved by polyacrylamide gel electrophoresis. Electrophoresis 2018, 39 (19), 2454-2459. 15. Harada, K.; Shiba, T.; Doi, K.; Morita, K.; Kubo, T.; Makihara, Y.; Piattelli, A.; Akagawa, Y., Inorganic polyphosphate suppresses lipopolysaccharide-induced inducible nitric oxide synthase (iNOS) expression in macrophages. PLoS One 2013, 8 (9), e74650. 16. Seidlmayer, L. K.; Gomez-Garcia, M. R.; Shiba, T.; Porter, G. A., Jr.; Pavlov, E. V.; Bers, D. M.; Dedkova, E. N., Dual role of inorganic polyphosphate in cardiac myocytes: The importance of polyP chain length for energy metabolism and mPTP activation. Arch. Biochem. Biophys. 2019, 662, 177-189. 17. Shiba, T., Inorganic polyphosphate and its chain-length dependency in tissue regeneration including bone remodeling and teeth whitening. In Inorganic Polyphosphates in Eukaryotic Cells, Kulakovskaya, T.; Pavlov, E.; Dedkova, E. N., Eds. Springer International Publishing: Cham, 2016; pp 139-158. 18. Tijssen, J. P.; Dubbelman, T. M.; Van Steveninck, J., Isolation and characterization of polyphosphates from the yeast cell surface. Biochim. Biophys. Acta 1983, 760 (1), 143-8. 19. Chen, K. Y., Study of Polyphosphate Metabolism in Intact Cells by 31-P Nuclear Magnetic Resonance Spectroscopy. In Inorganic Polyphosphates: Biochemistry, Biology, Biotechnology, Schröder, H. C.; Müller, W. E. G., Eds. Springer Berlin Heidelberg: Berlin, Heidelberg, 1999; pp 253-273. 20. Lindner, S. N.; Vidaurre, D.; Willbold, S.; Schoberth, S. M.; Wendisch, V. F., NCgl2620 encodes a class II polyphosphate kinase in Corynebacterium glutamicum. Appl. Environ. Microbiol. 2007, 73 (15), 5026-33. 21. Pilatus, U.; Mayer, A.; Hildebrandt, A., Nuclear polyphosphate as a possible source of energy during the sporulation of Physarum polycephalum. Arch. Biochem. Biophys. 1989, 275 (1), 215-23. page 12 of 19


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505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531


22. Ugochukwu, E.; Lovering, A. L.; Mather, O. C.; Young, T. W.; White, S. A., The crystal structure of the cytosolic exopolyphosphatase from Saccharomyces cerevisiae reveals the basis for substrate specificity. J. Mol. Biol. 2007, 371 (4), 1007-21. 23. Wurst, H.; Kornberg, A., A soluble exopolyphosphatase of Saccharomyces cerevisiae. Purification and characterization. J. Biol. Chem. 1994, 269 (15), 10996-11001. 24. Robbins, P. W.; Lipmann, F., Enzymatic synthesis of adenosine-5'-phosphosulfate. J. Biol. Chem. 1958, 233 (3), 686-90. 25. Beutler, E.; Kuhl, W., Characteristics and significance of the reverse glucose-6-phosphate dehydrogenase reaction. J. Lab. Clin. Med. 1986, 107 (6), 502-7. 26. Drake, H. L.; Goss, N. H.; Wood, H. G., A new, convenient method for the rapid analysis of inorganic pyrophosphate. Anal. Biochem. 1979, 94 (1), 117-20. 27. Van Wazer, J. R., Phosphorous and its compounds. Volume I: Chemistry. Interscience publishers, Inc.: New York, 1958. 28. Raj, G., Advanced inorganic chemistry Vol. I. 31 ed.; Krishna Prakashan Media: Meerut India, 2008. 29. Smith, S. A.; Baker, C. J.; Gajsiewicz, J. M.; Morrissey, J. H., Silica particles contribute to the procoagulant activity of DNA and polyphosphate isolated using commercial kits. Blood 2017, 130 (1), 8891. 30. Nyren, P.; Lundin, A., Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis. Anal. Biochem. 1985, 151 (2), 504-9. 31. Lundin, A., Optimization of the firefly luciferase reaction for analytical purposes. Adv. Biochem. Eng. Biotechnol. 2014, 145, 31-62. 32. Kowalczyk, T. H.; Phillips, N. F., Determination of endopolyphosphatase using polyphosphate glucokinase. Anal. Biochem. 1993, 212 (1), 194-205. 33. Christ, J. J.; Blank, L. M., Analytical polyphosphate extraction from Saccharomyces cerevisiae. Anal. Biochem. 2018, 563, 71-78.

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532 533

534 535 536 537 538 539

Figures

Figure 1. Mechanism of the enzyme assay. The present method for the determination of the average polyP chain length is divided into assays for the quantification of the total polyP concentration (A) and the polyP chain concentration (B). The combination of both values can be used to calculate the average polyP chain length. Individual reactions (reaction 1-3, reaction 5-8) and the overall reactions (reaction 4 and 9) are depicted. Red arrows indicate cascade reactions.

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Page 14 of 23

Page 15 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


average polyP chain length for polyP with n ≥ 3

square brackets denote the peak area; z is either 1, 2 or 3, whichever signal is best resolved

Eq. 1

PP1 + PP2 + PP3 + [PP4] =2∗ [PPz]

Eq. 2

average polyP chain length P−subunits =

Eq. 3

total polyP minus blank µM Pi = total polyP µM Pi − blanktotal polyP [µM Pi ]

Eq. 4

Eq. 5

total polyP concentration minus blank [µM Pi ] polyP chain concentration minus blank [µM polyP chains]

polyP chain concentration minus blank [µM polyP chains] = 0.5 * (polyP2 after scPpx1p treatment [µM polyP2, as Pi ] - blank [µM polyP2, as Pi i])

optimal polyP concentration before scPpx1p treatment [µM] = 2.5 * chain length [P-subunits] average polyP chain length for polyPn

Eq. 6

=

2 ∗ relative amount polyP + average polyP chain lengthpolyP

∗ relative amountpolyP n≥3 n≥3 relative amount polyP + relative amountpolyP n≥3 2

2

540 541

Eq. 7

D-luciferin + ATP + O2

firefly luciferase

oxyluciferin + polyP2 + AMP + CO2 + light

Figure 2. Equations used in this study.

page 15 of 19



A) chain concentration 

dilute sample to 1 – 10 (best: 5) µM polyP2



pipette 100 µl sample in black microtiter plate



add 50 µl polyP2 detection reagent with 33,000 U scPpx1p, 0.075 U ATP sulfurylase, 0.075 U hexokinase, 0.075 U glucose 6phosphate dehydrogenase incubate 1 h at room temperature



B) chain concentration blank  dilute sample to 1 – 10 (best: 5) µM polyP2 equivalent  same as A 

add 50 µl polyP2 detection reagent with 0.075 U hexokinase and 0.075 U glucose 6phosphate dehydrogenase



same as A

C) total polyP concentration  dilute sample to 5 – 200 (best: 100) µM total polyP  pipette 100 µl sample in transparent microtiter plate  add 50 µl enzyme reaction buffer with 11,000 U scPpx1p and 0.013 U scIpp1p

D) total polyP concentration blank  same as C

  



542 543 544 545

read fluorescence (ex. 340/9 nm, em. 460/9 nm), which indicates the NADPH concentration



same as A

Page 16 of 23



incubate 1 h at 37°C add 50 µl Pi detection reagent incubate 2 min at room temperature read absorbance at 882 nm, which indicates the Pi concentration



same as C



add 50 µl enzyme reaction buffer without enzyme

 

same as C same as C



same as C



same as C

Figure 3. Stepwise protocol for the enzyme assay. The chain concentration is determined in A (measuring value) and B (blank). The total polyP concentration is quantified in C (measuring value) and D (blank).

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546 547 548 549 550 551 552 553 554 555 556 557 558


Tables Table 1. Interference of varying substances on the enzyme assay. PolyP m100 (average polyP chain length by enzyme assay: 54 P-subunits) was spiked in with the interfering substances, whose concentration is displayed in column one (DNA and RNA as monomer). The chain length of polyP m100 made it possible that the same total polyP concentration (150 µM) was used for both the determination of the total polyP concentration and the chain concentration. To illustrate the degree of contamination, column two shows the concentration of the interfering substances as the mass ratio “gram interfering substance to 1 g of polyP”. The total polyP concentration and the polyP chain concentration were determined with the standard enzyme assay. The average polyP chain length was calculated from these two concentrations. The mean interference from one experiment with three replicate measurements per substance concentration combination is reported in comparison to the control without the interfering substance. The following monomer molecular weights were assumed: 103 g * mol-1 for polyP m100, 327 g * mol-1 for DNA, and 339 g * mol-1 for RNA. Abbreviations: n. c., not calculable, because the chain concentration was not measured.

interfering substance concentration mass [g] to 1 g [µM] analyte

100,000 400,000 750,000

378.3 1,513.0 2,836.9

total polyP concentration

interference [%] polyP chain concentration

NaCl -2 2 2

polyP chain length

0 1 -10

-2 1 13

0 5 2

0 0 2

0.8 8.3 83.0

ADP 1 3 -96

-1 blank too high blank too high

2 n. c. n. c.

20 200 2,000

0.4 4.2 42.3

DNA -3 1 -21

-2 3 blank too high

-1 -2 n. c.

20 200 2,000

0.4 4.4 43.9

RNA -2 2 -11

4 6 12

-5 -4 -21

NaSO4 10 100 1,000

30 300 3,000

0.1 0.9 9.2

1 5 4

559

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560 561 562

Table 2. Chain length determination of seven polyPs with the enzyme assay and 31P NMR. For the enzyme assay, the experimental design was 2 x 7 x 3 (number of independent experiments done on separate days x number of tested polyPs x number of replicate measurements). For 31P NMR, the experimental design was 1 x 7 x 1.

polyP

mean chain length ± standard error of the mean enzyme assay

563

Page 18 of 23

polyP2 1.9 ± 0.0 polyP3 2.9 ± 0.0 mode 155 6.0 ± 0.2 Budit 4 20.3 ± 0.1 p700 53.1 ± 1.1 p100 54.2 ± 0.8 mode 385 274.3 ± 5.3 a determined with 31P NMR

31P

NMR

2.0 2.9 5.6 17.0 50.4 44.1 297.3

deviation between methods [%] 4 3 -6 -16 -5 -19 8

cyclic polyP [relative to linear polyphosphate]a

page 18 of 19

0.00 0.02 0.83 6.81 1.53 6.35 0.29


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564 565


Graphical abstract

566

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24


Page 20 of 23

average with n ≥ 3Chemistrysquare brackets denote the peak area; Page 21 of 23 polyP chain length for polyP Analytical Eq. 1

1 2 2 Eq. 3 4 Eq. 3 5 6 7 Eq. 8 4 9 10 115 Eq. 12 13 14 Eq. 6 15 16 17 Eq. 187

=2∗

z is either 1, 2 or 3, whichever signal is best resolved

PP1 + PP2 + PP3 + [PP4] [PPz]

average polyP chain length P−subunits =

total polyP concentration minus blank [µM Pi ] polyP chain concentration minus blank [µM polyP chains]

total polyP minus blank µM Pi = total polyP µM Pi − blanktotal polyP [µM Pi ]

polyP chain concentration minus blank [µM polyP chains]

= 0.5 * (polyP2 after scPpx1p treatment [µM polyP2, as Pi ] - blank [µM polyP2, as Pi i])

optimal polyP concentration before scPpx1p treatment [µM] = 2.5 * chain length [P-subunits] average polyP chain length for polyPn =

2 ∗ relative amount polyP + average polyP chain lengthpolyP ∗ relative amountpolyP n≥3 n≥3 2 relative amount polyP + relative amountpolyP ACS Paragon Plus Environment n≥3 2

D-luciferin + ATP + O2

firefly luciferase

oxyluciferin + polyP2 + AMP + CO2 + light

A) chain concentration dilute sample to 1 – 10 (best: 5) µM polyP2

1 pipette 100 µl sample in 2 black microtiter plate 3 4 add 50 µl polyP2 detection reagent with 5 33,000 U scPpx1p, 6 0.075 U ATP sulfurylase, 7 0.075 U hexokinase, 8 0.075 U glucose 6phosphate 9 dehydrogenase 10 incubate 1 h at room 11 temperature 12 13 14 read fluorescence 15 (ex. 340/9 nm, 16 em. 460/9 nm), which indicates the NADPH 17 concentration

B) chain concentration C) total polyP Analytical Chemistry blank concentration dilute sample to 1 – 10 dilute sample to 5 – 200 (best: 5) µM polyP2 (best: 100) µM total equivalent polyP same as A pipette 100 µl sample in transparent microtiter plate add 50 µl polyP2 add 50 µl enzyme detection reagent with reaction buffer with 0.075 U hexokinase and 11,000 U scPpx1p and 0.075 U glucose 60.013 U scIpp1p phosphate dehydrogenase

same as A

incubate 1 h at 37°C add 50 µl Pi detection reagent incubate 2 min at room temperature same as A read absorbance at 882 nm, which indicates ACS Paragon Plus Environment the Pi concentration

D) total polyP Pageblank 22 of 23 concentration same as C

same as C

add 50 µl enzyme reaction buffer without enzyme

same as C same as C same as C same as C

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