Page 49 - The Biochemistry of Inorganic Polyphosphates

March 9, 2004
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Enzymatic methods 33
(a) (b) (c) (d)

Figure 2.11 Electrophoresis of PolyPs with average chain lengths of
(a) 15, (b) 25, (c) 45 (Sigma) and (d) 188 (Monsanto) phosphate residues
in20 % polyacrylamide gel (PAAG)with 7Murea; stainingwas achieved
by using toluidine blue (Pestov et al., 2003).

Elaboration of new electrophoretic methods for PolyP separation is continuing. For
example, capillary electrophoretic separations of sodium PolyPs with chain lengths of
5 to 44 has been reported. In this work, a buffer containing pyromellitic acid, triethanolamine
and hexamethonium hydroxide gives high-resolution separation of linear and cyclic PolyPs
(Stover, 1997; Wang and Li, 1998).
Because of its efficiency, the electrophoretic method is now widely used in studies of
PolyPs. It should be noted that for electrophoretic evaluation, the PolyPs must be extracted
from biological material, while nucleic acids, proteins and other interfering compounds
must be eliminated.

2.9 Enzymatic Methods

The greatest advantage of enzymatic methods for PolyP determination is their high speci-
ficity to PolyPs. Their wide application in recent years results from the development of
adequate methods of obtaining PolyP-dependent enzymes in sufficient quantities.
The first method of enzymatic PolyP assay was proposed by Clark et al. (1986). In
this technique, PolyPs were determined by polyphosphate glucokinase obtained from Pro-
pionibacterium shermanii. Glucose-6-phosphate dehydrogenase reduced NADP through
utilization of the formed glucose-6-phosphate, and the increase in NADPH concentration
was measured.
At the present time, many methods using PolyP-dependent enzymes for the assay of their
substrates have now been developed. Polyphosphate kinase (PPK) catalyses the reversible