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

spectrophotometric method reported here has sensitivity comparable
to that of enzymatic methods but, in contrast, is inexpensive and less
time-consuming. Furthermore, it avoids the competitive inhibition
by orthophosphate in the enzymatic reactions coupled to the
hydrolysis of PPi.

Interference by SDS

The interference produced by the presence of SDS in the medium
was studied. Final concentrations of 0.50%, 1.0%, 5.0% and 10%
w/v were assayed. No statistically significant differences were found
(P > 0.05) up to 5% SDS upon comparison with the values observed
without SDS (Fig. 8). The absence of interference by SDS allows
working with a high number of samples without immediately
processing them, and also permits measuring PPi in cell extracts.

Interference by proteins

Interference by protein at final concentrations of 0.1, 1.0 and
2.5 mg/mL was studied using bovine serum albumin. The presence
of protein quenches the change in the A280 of Cu(II), possibly due to
the reaction of the lone electron pair on the nitrogen of the peptide
bond with Cu(II). For protein concentrations below 1 mg/mL, the
behaviour of the system is restored in the presence of 1% SDS
(Fig. 8). For concentrations of protein above 1 mg/mL, the addition
of 1% SDS does not restore the behaviour of the system, although it
does decrease the intensity of the interference. In these cases, protein
can be removed to eliminate the interference by precipitation with
trichloroacetic acid prior to the addition of CuSO4, as described
under material and methods. This treatment does not affect the
determination of PPi with CuSO4.

Interference by EDTA

Interference by EDTA at final concentrations of 0.1, 0.5 and 1
mM was studied. In all the cases, a decrease in the absorbance of

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