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M. FE DE LA TORRE Y COLS. AN. R. ACAD. NAC. FARM.

commonly determined by following the progress of enzymatic
activities, coupling the hydrolysis of PPi either to the oxidation of
NADH (13), to the emission of light by a luciferase (14) or to the
formation of certain purines (15).

The PPi anion forms complexes with metallic cations, mainly
divalent ones. This process is rapid and thermodynamically favoured.
Accordingly, PPi is often used for the extraction and quantification
of cations in soils (16), and for stabilizing Cu in plating and the
manufacture of printed circuits (17). Likewise, PPi interference in
the colorimetric determination of orthophosphate can usually be
avoided by forming complexes with Cu or nickel (18).

We have extensively characterised the spectrophotometric
changes in the Cu(II):PPi system, and modelled the absorption shift
as a function of the interactions of the involved chemical species. On
this basis, we report a new method for the determination of PPi,
based on the modification of the Cu(II) absorption spectrum in the
presence of PPi due to the formation of complexes between these
two species. The method avoids most of the drawbacks usually
encountered in the usual ways of PPi determination; it is simple,
rapid and inexpensive, and allows the evaluation of enzymatic
activities with a sensitivity limit of 0.1 µmol of PPi in the assay
conditions.

EXPERIMENTAL PROCEDURES

Reagents and chemicals

All solutions were prepared with deionized water.
Copper sulphate pentahydrate, sodium hydroxide, hydrochloric
acid, trichloro acetic acid and glycine were purchased from
PANREAC (Barcelona, Spain).
Sodium dodecyl sulphate (SDS) was purchased from BIORAD
(Munich, Germany).
Sodium pyrophosphate decahydrate (3-[1,1-dimethyl-(2-
hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), sodium salt

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