Page 46 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
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Methods of polyphosphate assay
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an increase in the chain length of the PolyPs (Figure 2.9):

SP c
SP st
tg(α) = (2.2)
C
In the above equation, SP c is the total intensity (peak area) of signals from the middle
phosphate groups, SP st is the standard P i signal intensity, and C is the PolyP concentration,
expressed in mg P i per ml. The effect of reduction of the contribution of inner phosphate
groups to the total intensity of the P c signals is obviously associated with the primary and
secondary structures of PolyPs.
From the experimental data (Figure 2.9), the following equation was derived (where n c
is the number of monomeric phosphate groups in the PolyP molecule):

5.838n c
tg(α) = + 0.005n c (2.3)
1.664 + n c
The experimental data correspond well to the theoretical calculation of tg(α) obtained
from Equation (2.3). With regard to the quantitative determination of PolyP and PolyP chain
length by using NMR spectroscopy, it must be taken into account that the total intensity of
the middle phosphate groups is proportional to the concentration of each individual PolyPs
with a certain chain length, but no proportional correlation is observed during the transitions
between PolyPs with a small number of phosphate groups and PolyPs with a large number
of these groups. The contribution of PP4 groups to the total intensity of the peak decreases
with an increase in the PolyP chain length. By using Equations (2.2) and (2.3), the chain
lengths of PolyP samples may be defined more accurately.
The 31 P NMR spectroscopic technique was used for the detection and study of PolyPs
in different organisms, including bacteria (Navon et al., 1977b; Ferguson et al., 1978; Rao
et al., 1985; Suresh et al., 1985; Kjeldstad and Johnson, 1987; Kjeldstad et al., 1988;
Lawrence et al., 1998), yeast (Den Hollander et al., 1981; Greenfeld et al., 1987; Hola-
han et al., 1988; Bourne, 1990; Loureiro-Dias and Santos, 1990; Beauvoit et al., 1991;
Castro et al., 1995, 1999; Vagabov et al., 1998, 2000; Gonzalez et al., 2000; Trilisenko
et al., 2002), fungi (Yang et al., 1993; Pilatus et al., 1989; Hesse et al., 2002), al-
gae (Elgavish and Elgavish, 1980; Elgavish et al., 1980; Sianoudis et al., 1986; Lund-
berg et al., 1989; Bental et al., 1990), and protozoa (Moreno et al., 2000). PolyPs were
also observed by this method in soils (Adams and Byrne, 1989). One of the advan-
tages of NMR spectroscopy is the possibility of observing changes in the PolyP signals
of living cells. Such an approach is widely used and gives important information about
the PolyP dynamics under different conditions (Suresh et al., 1985; Zhang and Majidi,
1994).
PolyPs, which can be detected by NMR spectroscopy are called ‘NMR-visible’, and
represent a more mobile fraction of the total PolyP content. Lack of an ‘NMR-visible’
PolyP signal does not indicate the absence of PolyPs in a sample. Accurate values of the
chemical shifts of these signals depend on the pH and residual concentrations of divalent
cations in the extract (Pilatus et al., 1989).
The intensities of the signals in the study of PolyPs by using 31 P NMR spectroscopy
directly depend on the degree of PolyP binding with other structures and compounds of