Page 31 - The Biochemistry of Inorganic Polyphosphates

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METHODS OF

POLYPHOSPHATE ASSAY

IN BIOLOGICAL

MATERIALS

2.1 Methods of Extraction from Biological Materials

The earlier work on the isolation of PolyPs from the cells of living organisms usually
employed the same methods as those used for the extraction of nucleic acids. It was not until
1936 that MacFarlane (MacFarlane, 1936) proposed a specific method for the extraction and
fractionation of condensed phosphates present in cells. It was found that these phosphates
could be divided into two main fractions, i.e. one soluble in 5 % trichloroacetic acid (TCA)
and the other insoluble, and ever since then cellular condensed polyphosphates have been
divided into acid-soluble and acid-insoluble fractions.
Although most workers have used the same extractant, 5 % TCA (or occasionally
0.5 M HClO 4 ), to obtain the acid-soluble fraction, a variety of methods have been used
to isolate acid-insoluble condensed phosphates. The most common method of extraction
of acid-insoluble PolyPs from cells involves the use of dilute sodium hydroxide solu-
tion, pH 9–12 (MacFarlane, 1936; Belozersky, 1955; Belozersky and Kulaev, 1957; Mudd
et al., 1958; Krasheninnikov et al., 1968). Widely used methods for the extraction of acid-
insoluble PolyPs from biological materials are several variants of the method of Schmidt
◦
and Thannhauser, i.e. the use of 1 M potassium hydroxide at 37 C for various periods of
time (Schmidt and Thannhauser, 1945; Chaloupka and Babicky, 1957, 1958; Zaitseva et al.,
1959; Griffin et al., 1965; Griffin and Penniall, 1966).
In addition, some PolyP fractions can be extracted by hot solutions of acids, either 5 %
TCA (Wiame, 1947a; Belozersky, 1955; Belozersky and Kulaev, 1957; Bukhovich and

The Biochemistry of Inorganic Polyphosphates I. S. Kulaev, V. M. Vagabov and T. V. Kulakovskaya
C 2004 John Wiley & Sons, Ltd ISBN: 0-470-85810-9

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