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

(AMPSO), N,N-bis(2-hydroxyethyl) glycine (BICINE), 2-[N-
cyclohexylamino]ethane, sulfonic acid (CHES), albumin from bovine
serum (BSA), 4-morpholinepropanesulfonic acid, sodium salt
(MOPS), sodium tetraborate, ethylenediamine-tetraacetic acid
(EDTA), adenosine 5'-triphosphate, disodium salt hydrate (ATP) and
DOWEX ® 50WX2-200 ion-exchange resin were purchased from
SIGMA-ALDRICH (Sant Louis, USA). Before use, the resin was
washed sequentially with 2 N sodium hydroxide, 2 N hydrochloric
acid and water, and then allowed to dry overnight.

Tris-(hydroxymethyl)aminomethane (TRIS) and sodium
hydrogenophosphate were purchased from PROLABO (Asturias,
Spain).

Magnesium chloride hexahydrate was purchased from PROBUS
(Barcelona, Spain).

Instruments

A Beckman DU-640 spectrophotometer with cuvettes
thermostated at 37 ºC was used to measure absorbance at 280 nm
(A280). Beckman quartz microcuvettes with 10 mm pathlength,
700 µl capacity and a 300 µl minimum volume for reading were used.

Procedure

Blank: 50 µL 10 mM CuSO4, 100 µl 0.5 M AMPSO, 100 µl 20%
SDS, 200 µl 50 mM Na4P2O7 and 550 µl H2O. Final concentrations
were: [Cu]tot = 0.5 mM and [PPi]tot = 10 mM. Sample: 50 µl 10 mM
CuSO4, 100 µl 0.5 M AMPSO, 100 µL 20% SDS, 500 µl of a solution
containing PPi and 250 µl H2O. The value of A280 for the sample can
be used directly for reading the concentration of PPi in the
calibration curve (Fig. 7) or for calculating it by means of the
corresponding expression (equation 1). If the concentration of
protein in the sample was higher than 1 mg/mL, the protein was
eliminated by precipitation with trichloroacetic acid before the
addition of CuSO4. If the concentration of Mg(II) in the sample was
higher than 1 mM, it was removed by the addition of DOWEX-50 at

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