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VOL. 72 (3), 463-487, 2006 SPECTROPHOTOMETRIC CHARACTERISATION OF THE...

An enzymatic method for measuring PPi coupled to NADH

oxidation is commercially available, but is expensive: each

determination has an average cost of 10€ and needs ca 30 min per
point to be completed. Compared with this, Cu(II) titration is

inexpensive and the only additional reagent is inorganic, CuSO4, thus
allowing low assay costs.

CONCLUSIONS

Here we present a comprehensive chemical and
spectrophotometric description of the Cu(II):PPi, implementable
as spectrophotometric method for the quantification of PPi. This
method is based on modification of the Cu(II) absorption spectrum
in the presence of PPi by following the formation of complexes
between these two species. The sensitivity limit of the method is
0.1 µmol of PPi, such that it is possible to titrate small amounts of
this product and work with microsamples.

The determination is rapid. The formation of complexes between
Cu(II) and PPi is immediate and these complexes are stable at the
assay temperature, so it is possible to analyse many samples in a
short period of time.

The procedure to quantify PPi is also simple and a general
procedure is described including preparation of the blank and the
sample at the assay pH. SDS is added to avoid interferences by
proteins, nucleotides, EDTA and Mg(II). Modifications of the method
in the presence of high concentrations of proteins and Mg(II) do not
represent a significant change in this general procedure and are also
properly detailed.

The determinations achieved with the proposed method are
inexpensive. The only additional reagent is CuSO4, and hence the
assays can be considered to have virtually zero cost.

These characteristics make the Cu(II) titration appropriate for
the determination of PPi in chemical and biochemical reactions. The
method also allows the measurement of PPi in the presence of
orthophosphate. Accordingly, it is possible to quantify mixes of these
two analytes.

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