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 HCIO4), 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 solution, 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 etal., 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 

Belozersky, 1958,1959 Konovalov, 1960) or 10 % perchloric acid, at 80-100 °C (Krishnan etal, 1957 Drews, 1960b Fedorov, 1961 Harold, 1960,1962ab lames andCasida, 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 the author s opinion, this method ensures an almost complete extraction of acid-insoluble PolyPs from the cells of bacteria and other microorganisms. 

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