Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

8 Yazawa
DuPont-NEN and the molar concentration of it is calculated from the specification
data shipped with the radionuclide.
12. Protein solution: Contaminating Ca 2+ in the protein solution should be reduced to
less than 0.1 mol/mol of the total Ca 2+ -binding sites in the initial sample solution.
Several methods have been reported to reduce the contaminating Ca 2+ in the
neutral pH such as addition of 50–100 mM EDTA or ethyleneglycol-bis
(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (optionally in the presence
of 6 M urea) followed by passage through a Sephadex G-25 desalting column
(12), and extensive dialysis against or column chromatography with
Chelex-100 chelating resin (2). For stable proteins such as calmodulin, troponin
C, and parvalbumin, addition of trichloroacetic acid to 3% precipitates Ca 2+ -free
proteins, which can be renatured by neutralization (6,13). Precipitates were collected
by centrifugation, homogenized with addition of small volume of deionized water,
and dissolved with addition of small volume of 2 M Tris base, which is followed
by passage through a Sephadex G-25 column. The concentration of contaminating
Ca 2+ in the protein solution is determined by atomic absorption spectrometry, and
is taken into account in the calculation of the Ca 2+ -binding data.
Protein concentrations must be determined with a suitable and reliable
method, such as quantitative amino acid analysis or UV spectro-photometric
determination.
3. Methods
The following procedures, except for the final calculation step, as well as
the dilution of 45 Ca 2+ aforementioned, are to be carried out in a room equipped
with facilities necessary for handling of radioisotopes.
3.1. Assembly of the Flow-Dialysis Cell
When everything is ready to begin, the flow-dialysis cell, which has been
stored as disassembled parts, is assembled with mounting a dialysis membrane
(see Fig. 3) (see Note 6).
1. Immerse the degassed buffer solution in the 500 mL Erlenmeyer flask in the water
bath and equilibrate at 25°C.
2. Fill a disposable plastic syringe (5 mL) with the buffer solution and, after
removal of air bubbles, connect it to the PE tube (inlet) of the buffer chamber of
the dialysis cell.
3. Fill the buffer chamber with the buffer solution sent from the syringe through the
PE tube.
4. Fill the other PE tube (outlet) with the buffer solution from the chamber by sucking
with another syringe connected to the other end of PE tube.
5. Put a magnetic stirring bar (discoid, 1 cm in diameter) into the chamber, and fill
the chamber with the buffer solution.