Enzymatic quantification and length determination of polyphosphate down to a chain length of two
Graphical abstract
Introduction
Inorganic polyphosphate (polyP) can be found as a linear polymer of orthophosphate (Pi) in all living organisms [1]. The chain length can range from two up to several thousand P subunits [2,3]. Sensitive, specific and robust methods for the quantification of polyP are required to study P physiology in living organisms. One of the most popular enzymes used for polyP quantification is the Saccharomyces cerevisiae exopolyphosphatase 1 (scPpx1p). ScPpxp1p was first purified and characterized by Wurst and Kornberg [4]. ScPpx1p hydrolyses one polyPn to n-2 Pi and one pyrophosphate (PPi) (reaction 1).(n - 2) H2O + polyPn → (n-2) Pi + PPi
Since its discovery scPpx1p has been utilized to quantify polyP by enzymatic hydrolysis of polyP and subsequent Pi quantification by a colorimetric assay [[5], [6], [7], [8], [9], [10], [11], [12]]. Because polyP hydrolyses under acidic conditions [13], high acidity and prolonged exposure during Pi detection must be avoided to prevent underestimation of polyP. The ascorbate and malachite green assays are the two most common colorimetric assays for the detection of Pi. In both assays one Pi spontaneously forms a complex with twelve molybdate (MoO42−) under acidic conditions [14]. In the ascorbate assay this complex is reduced by ascorbate to its blue form [15]. Antimony greatly accelerates the reduction [16]. During the malachite green assay the deprotonated form of malachite green binds to the molybdate phosphate complex giving it a green color [17]. The malachite green assay is more sensitive in terms of the lower limit of detection (1 nmol Pi) and the slope of the calibration curve (0.120 absorbance units per nmol Pi) than the ascorbate assay (limit of detection: 2 nmol Pi; slope: 0.037 absorbance units per nmol Pi), and can, therefore, be applied to matrices with very low Pi concentrations [14]. The malachite green assay and the antimony ascorbate assay require 20 to 30 [17] and fewer than 10 min for full color development [18], respectively. Due to the long incubation time, the malachite green assay can only be applied to matrices, where P hydrolysis is of little concern.
The two most common methods for polyP chain length determination are end group titration and polyacrylamide gel electrophoresis (PAGE). The end group titration is based on the fact that in polyP there is one strong hydrogen (Kdissociation ≈ 100 to 10−3) for each P atom as well as one weak hydrogen (Kdissociation ≈ 10−7 to 10−9) at either end of the chain. The combination of the strong and weak hydrogens results in inflection points in the titration curves of polyP at pH values near 4.5 and 10 [13]. The average chain length can be calculated from the consumption of hydroxyl ions during the titration of polyP from pH 4.5 to 9.5 and the total P content [19]. PAGE allows for the separation of polyP by size in polyacrylamide gels driven by electric current. High acrylamide concentrations (e. g., 20% (m/v)) and a denaturing agent (e. g., 7 M urea) allow for the separation of polyP chain lengths shorter than 100 P-subunits. After electrophoresis polyP is visualized by toluidine blue staining [20]. End group titration requires gram amounts of polyP and high purity. Both criteria are rarely met by biological samples. PAGE, on the other hand, demands only μg amounts of polyP, but yields only a qualitative chain length distribution. PolyP length standards allow even for semi-quantitative results. However, polyP length standards are difficult to obtain [20] and commercially not available. Nuclear magnetic resonance and Raman spectroscopy can also be used to semi-quantitatively gauge polyP chain length [21,22]. Both rely on specific instrumental analytics, which, unfortunately, are rarely available in the laboratory. Moreover, both methods do not allow for higher throughput. In conclusion, although of great interest to polyP research, a method for quantitative determination of polyP chain length from biological samples with little equipment is lacking.
It is apparent from reaction 1 that polyP cannot be fully detected, because after enzymatic hydrolysis with scPpx1p every polyP molecule yields one PPi, which is not detected by the Pi assay. Table 1 shows schematically that short chain length polyP cannot be measured with scPpx1p.
This underestimation is troublesome for polyP with an average chain length shorter than 100. Such short polyP can be found, for example, in S. cerevisiae vacuoles (length: < 25) [23] or mitochondria (length: < 15) [24], three from five fractions of the popular analytical polyP extraction used by Kulaev et al. applied to S. cerevisiae [10,11,22,25], sea lettuce (Ulva lactuca, length: < 20) [26], the algae Heterosigma akashiwo (length: < 20) [27], acidocalcisomes of Trypanosoma brucei (pathogen causing sleeping sickness), Trypanosoma cruzi (pathogen causing Chagas disease) and Leishmania major (pathogen causing leishmaniasis; length of all three: < 4) [28], and Toxoplasmoa gondii (pathogen causing toxoplasmosis, length from one of two polyP fractions: < 50) [29].
We hypothesized that the addition of the S. cerevisiae inorganic pyrophosphatase (scIpp1p), which hydrolyses PPi to Pi (reaction 2), will allow the comprehensive quantification of polyP including polyP with a chain length of two.PPi + H2O → 2 Pi
On top of this, the use of scIpp1p and scPpx1p should allow for the length determination of polyP. The proposed workflow includes as a first step separate hydrolysis of a sample without enzyme, with scPpx1p, and with both scPpx1p and scIpp1p. The second step contains the colorimetric Pi quantification. Hereby, the blank Pi, the scPpx1p digestible polyP and the total polyP can be measured, respectively. The average polyP chain length can be calculated by Equation (3). The blank Pi is defined as direct Pi quantification from the sample. The scPpx1p digestible polyP is defined as sample plus scPpxp1p, followed by Pi quantification. Total polyP is defined as the sample plus scPpx1p and scIpp1p, followed by Pi quantification. Both scPpx1p digestible polyP and total polyP include the blank Pi. The denominator of Equation (3) calculates the concentration of PPi in the sample. Dividing the numerator through the denominator yields the average chain length of the scPpx1p digestible polyP. The addition of two (i. e., the chain length of PPi) gives the overall average polyP chain length. For example, one molecule of polyP with a chain length of five subunits accompanied by one molecule of Pi (i. e., the Pi blank), would result in an average chain length = 2 * (4–1) * (6–4)−1 + 2 = 5.
The goal of this study was to develop an enzymatic assay for both comprehensive polyP detection, and determination of polyP chain length down to a chain length of two. The method was supposed to be based on differential enzymatic hydrolysis of polyP with scPpxp1p, and a mixture of scPpx1p and scIpp1p. After enzymatic polyP hydrolysis released Pi should be detected with a colorimetric Pi assay. The advantages and disadvantages of this enzymatic assay for polyP sizing in comparison to state of the art methods are discussed.
Section snippets
Chemicals
PolyPs Budit 4, 7, 9 were from Budenheim (Germany). The number indicates the pH of a 1% (w/v) solution (the longer the polyP, the more acidic it reacts). Sodium hexametaphosphate and trisodium trimetaphosphate were purchased from Sigma-Aldrich (MO, USA). Sodium tripolyphosphate (termed “triphosphate” henceforward) was from VWR (PA, USA). Salmon sperm DNA, RNA sodium salt, sodium diphosphate and sodium metaphosphate were acquired from Carl Roth (Germany). All reagents involved in Pi analysis
The ascorbate assay is superior to the malachite green assay for measuring Pi in the presence of polyP
One malachite green assay and one ascorbate assay were compared for the colorimetric detection of Pi. The Pi assay will later be used to measure the Pi blank and the Pi concentration after enzymatic hydrolysis of a sample. It is of utmost importance that the Pi assay does not hydrolyze polyP, because this would result in an underestimation of polyP concentrations. To minimize polyP hydrolysis during Pi detection, the acidity and incubation time during Pi detection should be kept to a minimum.
A
Optimal Pi detection after enzymatic polyP hydrolysis
The malachite green assay is one of the most frequently used assay to detect Pi after enzymatic polyP hydrolysis [6,8,12,33,34]. Van Veldhoven and Mannaerts first developed the malachite green assay and recommend an incubation period of 30 min after the addition of the detection reagents to the sample. Indeed, we can confirm this finding (compare Fig. 2A from this paper with Fig. 1 from Ref. [17]). It should be considered that polyP non-enzymatically hydrolyses in the acidic conditions of Pi
Conflicts of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Author contribution
JJC conducted the experiments. Data analysis and writing of the paper was done by LMB and JJC. LMB initiated and coordinated the study. All authors read and approved the final manuscript.
Acknowledgments
The Deutsche Bundesstiftung Umwelt (DBU) is gratefully acknowledged for providing financial support. We would like to thank Dr. Rainer Schnee and the company Chemische Fabrik Budenheim KG for suppling us with polyP standards. The authors would like to thank the anonymous reviewers for their helpful and constructive comments.
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