Page 33 - The Biochemistry of Inorganic Polyphosphates

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Chromatographic methods 17
specific physiological roles. PolyPs were most completely extracted from yeast cells by
this method (Table 2.1), whereas the method employed by Chernyshova et al. (1971) and
Clark et al. (1986) permitted the extraction of only about 80 % of PolyPs from biomass.
The sequential treatment of yeast cells with cold diluted perchloric acid, salt and weak
alkali allowed the isolation of PolyPs with degrees of polymerization from as low as 2 to
8 to as high as 200. Moreover, the former method (Kulaev et al., 1966a) made it possible (see
table 2.1) to isolate five PolyP fractions from yeast cells, whose synthesis and degradation
are closely related to metabolic processes in individual cell compartments. The extraction
of PolyP fractions depends rather on their state or localization in the cell than on the degree
of polymerization.
Clark et al. (1986) found that two different extractions were required to isolate all
PolyPs from Propionibacterium shermanii, yet these two fractions contained PolyPs of
identical size. These authors concluded that one fraction was soluble PolyP, while the
second fraction was more tightly complexed in granules. The most frequently used pro-
cedure, i.e. extraction with ice-cold trichloroacetic acid (TCA), followed by extraction
with alkali, and also the Langen and Liss variation of this procedure, do not extract all
PolyPs from P. shermanii or from several other organisms. To demonstrate that the chains
were not shortened by the extraction procedure developed for P. shermanii, Clark et al.
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(1986) included P-labelled PolyP during the extraction and then analysed the radioactive
PolyP before and after the procedure by using gel electrophoresis. They found that their
procedure did not cause PolyP hydrolysis, in contrast to the procedures of Langen and Liss
(1959) and Harold (1966). Other procedures that are apparently mild include extraction
with hot water, while sodium dodecylsulfate, carbon tetrachloride and phenol/chloroform
have been used to extract PolyPs which are apparently located in granules.
For the analysis of PolyPs in activated sludge, some modifications of the extraction
method have been developed. M¨ussig-Zufika et al. (1994) compared various chemical
fractionation methods to retrieve intact PolyPs from activated sludge and pure cultures.
They concluded that the degree of PolyP hydrolysis during the treatment was strongly de-
pendent on the extraction method being employed. Using the method of Mino et al. (1985)
(with cold TCA extraction), 24 % of the PolyPs were hydrolysed to P i , while only 5 % and
1 % hydrolysis occurred by the methods of Psenner et al. (1984) and Clark et al. (1986),
respectively. The ‘Clark method’, which included a cold TCA–acetone extraction step, was
not suitable for Gram-positive bacteria and for bacteria from activated sludge due to their
cell wall structures. M¨ussig-Zufika et al. (1994) described a modified extraction method
applicable to pure cultures, mixed cultures and activated sludge. This technique was essen-
tially a combination of various extraction methods (including mechanical agitation) and did
not result in PolyP hydrolysis, producing intact PolyP chains that could be analysed further.
It should be concluded that PolyP extraction from ‘new’ organisms, where PolyP
metabolism has been little studied, needs careful verification of the fullness and intact-
ness of the PolyP chains.

2.2 Chromatographic Methods

Reliable identification of polyphosphates often includes the ‘Thilo and Wicker method’ of
chromatographic analysis of the products of partial hydrolysis (Thilo and Wicker, 1957).
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In this approach, the hydrolysis of condensed phosphates in neutral solution at 60 C yields