Page 36 - The Biochemistry of Inorganic Polyphosphates

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Methods of polyphosphate assay
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0.5 1 2 3 4 5 6 7 8 9 10 11 12 0.5
mg PO 4 per fraction 0.3 KCl 0.3 [KCl](M)
0.4
0.4
0.2
0.2
0.1

10 20 30 40 50 60 70 80 90 100 110120 130 140 150160 170 180 0.1
Fraction

Figure 2.2 Separation of PolyPs of low molecular weight by ion-exchange chromatography on
Dowex 1 XI0 in a KCl gradient. The numbers 1–12 represent the number of phosphorus atoms in the
PolyP molecules constituting the various fractions (Matsuhashi, 1963).

high-molecular-weight PolyPs. The first of these is Van Wazer’s method for the fractional
precipitation of PolyPs of different chain lengths from aqueous solutions with acetone (Van
Wazer, 1958). This technique yields a great number of fractions, which differ from each
other in the lengths of the PolyP chains. The second method, also developed in the labora-
tory of Van Wazer (Ohashi and Van Wazer, 1964), gives similar results to those obtained
by paper chromatography. A method for the separation of inorganic PolyPs on Sephadex
columns has also been developed in Ebel’s laboratory (Felter et al., 1968). Gel filtration on
Sephadex G-10 is a suitable method for the purification of PolyP from P i and PP i (Andreeva
and Okorokov, 1993).
Some methods for fast chromatographic separation and detection of PolyPs in food, bio-
logical samples or water have been proposed (Halliwell et al., 1996; Baluyot and Hartford,
1996; Svoboda and Schmidt, 1997; Bewsler et al., 2001). A single-column chromatographic
system with indirect UV detection was elaborated, and the dependencies of PolyP retention
on the concentrations of pyromellitic acid and ethylenediaminetetraacetic acid (EDTA) in
the mobile phase and on the pH of the eluent were determined (Svoboda and Schmidt,
1997). A high performance liquid chromatography (HPLC) method has also been used for
the separation of PolyPs (Lorenz and Schr¨oder, 1999).

2.3 Colorimetric and Fluorimetric Methods

One of the simplest methods of estimation of PolyPs in extracts is based on the assay of
P i , which is released from the PolyPs by hydrolysis with 1 M HCl at 90 C for 10 min.
◦
The P i released under these conditions is defined as ‘labile phosphorus’. If the compounds
containing organic labile phosphorus (i.e. nucleotide phosphates, sugar phosphates, etc.)
were removed from the extracts by adsorption on Norit charcoal, the increase in P i content
after hydrolysis can be attributed to PolyP and pyrophosphate (PP i ). Estimation of the PP i
content (Mansurova, 1989) before hydrolysis may be needed in some cases for more precise
calculations of the PolyP content. P i may be determined by one of the well-known chemical
methods (Fiske and Subarrow, 1925; Weil-Malerbe and Green, 1951).