Page 32 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-02
Methods of polyphosphate assay
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Belozersky, 1958, 1959; Konovalov, 1960) or 10 % perchloric acid, at 80–100 C (Krishnan
et al., 1957; Drews, 1960b; Fedorov, 1961; Harold, 1960, 1962ab; James and Casida, 1964).
When this method of extraction was employed, the condensed phosphates are hydrolysed
to orthophosphate, the amount of which indicates the amount of condensed phosphates
present in the acid-insoluble fraction. Hughes and co-workers (Hughes et al., 1963) used
a prolonged (5 h) extraction of acid-insoluble PolyPs with 10 % TCA at 20–22 C. In
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the author’s opinion, this method ensures an almost complete extraction of acid-insoluble
PolyPs from the cells of bacteria and other microorganisms.
Later investigations showed that it was possible to carry out further fractionation of the
PolyPs present in biological material depending on the chain length. Such fractionation
has been carried out by Langen and Liss in the laboratory of Lohmann (Langen and Liss,
1958ab, 1959; Liss and Langen, 1960, 1962). Their method consists of successive extraction
of cells in the cold with 1 % TCA, a saturated solution of a salt such as NaClO 4 , dilute NaOH
solution (pH 10), and a more concentrated solution of alkali (0.05 N NaOH). This method,
either in its original version or modified in various ways, has been used extensively for the
fractionation of PolyPs from different organisms. Its advantage is that the fractions obtained
were localized at different intracellular sites and showed different physiological activity.
Another, and apparently successful, method for the fractionation of PolyPs present in
biological material is that developed by Miyachi and co-workers (Miyachi, 1961; Miy-
achi and Tamiya, 1961; Miyachi and Miyachi, 1961; Miyachi et al., 1964). These resear-
chers successively extracted the PolyPs present in the cells of Chlorella and other
organisms with 8 % TCA in the cold (fraction A), then with a solution of NaOH,
pH 9, in the cold (fraction B), and finally with a 2 N solution of KOH at 37 C for 18 h.
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The PolyPs extracted with 2 N KOH by the method of Schmidt and Thannhauser were
further separated by Miyachi into two fractions, i.e. one precipitated by neutralization with
HClO 4 in the presence of KClO 4 (fraction C) and that which was not precipitated under
these conditions (fraction D). The work of Miyachi showed that this mode of fractionation
of Chlorella PolyPs yielded fractions which differed in their physiological activity and also
apparently cellular location.
It should be pointed out that neither of the methods described above was aimed at
obtaining completely unmodified preparations of cellular condensed polyphosphates. Other,
much milder, methods of extraction from cells have been developed in order to obtain
samples of condensed phosphates, which are as little modified as possible to completely
avoid the use of strong acids and alkali.
The mildest methods for the extraction of condensed phosphates are as follows:
(i) extraction with hot water (Kornberg and Kornberg, 1954; Chayen et al., 1955; Lohmann
and Langen, 1956; Dirheimer and Ebel, 1957; Dirheimer, 1964); (ii) extraction with dilute
sodium carbonate solution (Ingelman and Malmgren, 1950; Ebel, 1952a,b); (iii) extrac-
tion with hot 2 M sodium chloride solution (Kaltwasser and Schlegel, 1959; Kaltwasser,
1962; Kaltwasser et al., 1962); (iv) extraction with cold distilled water following a prelim-
inary treatment of the material with alcohol and ether (Schmidt et al., 1946; Malmgren,
1949; Ebel, 1952a; Lohmann and Langen, 1956; Dirheimer and Ebel, 1957; Chaloupka and
Babicky, 1958; Dirheimer, 1964); (v) extraction with sodium hypochlorite (Harold, 1963b).
On the basis of the work carried out at our laboratory, we consider the extraction
method of Langen and Liss (1959) with the modification of Kulaev et al. (1966a) to
be one of the best available for the separation and quantitative determination of dif-
ferent PolyP fractions localized at different intracellular sites and apparently displaying