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

based on the generation of ammonium phosphomolybdate (9), is its
selectivity: Cu(II) titration allows measurement of 0.5 mM PPi in the
presence of 50 mM orthophosphate.

The spectrophotometric method reported here is especially useful
for quantifying PPi in the biochemistry laboratory. In this
environment, enzymatic methods are the standard, but they fail to
achieve direct detection of PPi; instead, they follow the progress of
enzymatic activities such as the oxidation of NADH (13), the emission
of light by a luciferase (14) or the formation of purines (15), all of
them coupled to the hydrolysis of PPi. With the titration with Cu(II)
it is possible to measure PPi directly, therefore decreasing the error
in the measurements. Regarding sensitivity, the proposed method is
more sensitive than following NADH oxidation, similar to the
monitoring of purine synthesis, and less sensitive than quantification
of light emission by luciferase. Before applying these enzymatic
methods, moreover, the activities of many reagents must be
monitored to ensure that PPi is in fact the rate-limiting component
in the assay. Furthermore, the components of such assays often
contain compounds that may hamper the proper development of
enzyme activity. It is necessary therefore to spend time establishing
the optimum assay conditions before using these methods (22).
Additionally, enzymatic protocols are usually cumbersome, they
waste reagents and are time-consuming. In comparison with this, the
method here described is rapid and these complexes are stable at the
assay temperature used.

Regarding the robustness of the assay, the use of SDS makes it
possible to stop enzymatic reaction and collect a large number of
samples or cell extracts with no need of immediately reading each of
them. Interferences by proteins up to 1 mg/mL, EDTA up to 1 mM,
ATP up to 5 mM and MgCl2 up to 1 mM can be overcome by addition
of SDS to the sample. Regarding the presence of nucleotides,
enzymatic methods such as that based on the formation of purines
permit the presence of ATP only up to 0.5 mM; others, such as that
employing luciferase, cannot be used if ATP is a component of the
sample. Simple modifications of the general procedure are also
provided for cases with higher concentrations of proteins or MgCl2
in the sample.

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