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

interference to be controlled up to a concentration of 5 mM ATP
(data not shown).

Interference by Mg(II)

The presence of Mg(II) is essential for the catalytic activity of
many enzymes whose reaction product is PPi. Accordingly,
interference by Mg(II) at concentrations of 1.0, 5.0 and 10.0 mM was
studied. It resulted in a decrease in the absorbance of Cu(II) that
was proportional to the concentration of MgCl2 in the sample,
probably due to the competition between Mg(II) and Cu(II) for PPi.
The presence of 2% SDS allowed this interference to be overcome up
to a concentration of 1 mM Mg(II) (data not shown). For higher
concentrations of Mg(II), it was necessary to remove this cation
from the reaction medium. This was accomplished by the addition
of DOWEX-50 at the pH of the assay (AMPSO/NaOH pH 9.4),
followed by centrifugation. The interference is thus abolished and
the supernatant can then be treated as formerly described.

DISCUSSION

The interaction of PPi with Cu(II) in water solution modifies the
absorption spectrum of the cation. We have spectrophotometrically
characterised the Cu(II):PPi system and modelled the interaction
between the different chemical species. We have provided here
a proper description of the system, the equilibria, and the
spectrophotometrical behaviour of the absorbent species. Since the
changes in absorptivity are concentration-dependent, we have
implemented the knowledge of the system into a spectrophotometric
method for determining PPi in solution. The main advantages of the
method for PPi determination here presented are its speed, simplicity,
price, and relative high sensitivity. In comparison with methods based
on chromatography (10) or capillary electrophoresis (11), titration
with Cu(II) is more sensitive and the procedure is simpler, regarding
both sample preparation and data reading.

Methods based on chemical reactions are less sensitive than the
assay reported here. Another important difference with the method

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