Page 47 - The Biochemistry of Inorganic Polyphosphates

15:25
Char Count= 0
March 9, 2004
WU095-02
WU095/Kulaev
Gel electrophoresis 31
the cell, in particular, with metal ions. Therefore, while the detection of a PolyP signal
indicates the presence of these compounds in the cell, the absence of this signal still cannot
be considered as direct evidence for the absence for PolyPs, as was well described in the
work using Chlorella fusca (Sianoudis et al., 1986).
In some microorganisms of aerobically activated sludge, the NMR resonance character-
istics of PolyPs were only observed when the cell structure was disrupted by treating with
a strong alkali (Pereira et al., 1996).
31
The interaction with cations lays the basis for the P NMR spectroscopic method, allow-
ing one to distinguish between extracellular and intracellular pools of PolyPs. Ethylenedi-
aminetetraacetic acid (EDTA) can be used to complex the divalent cations bound to PolyPs
31
and to produce a new P NMR shift. However, because the cell membrane is impermeabile
to EDTA, only extracellular PolyPs is affected. This method was used successfully on a cell
suspension of Propionibacterium sp. (Serafim et al., 2002).
Thus, authors often apply the term ‘visible’ PolyPs when discussing the results ob-
tained by this method (Loureiro-Dias and Santos,1990). It can be considered that the
most reliable approach is the combination of PolyP chain length determination by NMR
spectroscopy with the method of chemical extraction from cells and their quantitative
analysis.
The above approach allowed determination of the content and polymerization degree of
PolyPs in volutin granules (Jacobson et al., 1982), in vacuoles (Trilisenko et al., 2002), and
in different PolyP fractions obtained from yeast cells (Vagabov et al., 1998, 2000). As an
example, Figure 2.10 shows the 31 P NRP spectra of the acid-soluble PolyPs from isolated
yeast vacuoles.
Thus, the NMR approach gives a most precise picture of the PolyP content and
polymerization degree in different cell compartments by a combination of NMR spec-
troscopy with the methods of sub-cellular fractionation and chemical extraction of
PolyPs.

2.7 Other Physical Methods

Infrared spectroscopy has been rarely used for PolyP characterization (Datema et al., 1977).
Electrospray ionization mass spectrometry (ESI–MS) has been applied to the characteriza-
tion of phosphates (P i ,PP i , PolyP 3 , PolyP 4 , and tricyclophosphate). The high selectivity of
ESI–MS allowed the detection of these compounds without any pre-separation by ion chro-
matography or capillary electrophoresis. The limits of detection for ESI–MS were estimated
to be in the range from approximately 1 to 10 ng ml −1 (Choi et al., 2000).

2.8 Gel Electrophoresis

One of the most effective and widely used methods for PolyP separation is gel electrophore-
sis. This technique was adapted for PolyPs by Robinson et al. (1984, 1987) and Pepin et al.,
1986. Electrophoresis in 15–20 % polyacrylamide gel allows PolyP resolution up to a chain
length of 100–200 phosphate residues. A mixture of 90 mM trisborate (pH 8.3) with 2.7 mM