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Immo bilked Metal Ion Interaction 25
3.2.2. pH Gradient Elution
(Continuous Buffer System)
1. After sample application, elute with 2.5 bed vol of 20 mM sodium
phosphate, 0.5M NaCl, pH 7.
2. Start a linear pH gradient of 20 mM sodium phosphate, 0.5M NaCl,
pH 7 to 50 mM sodium phosphate, 0.5MNaC1, pH 4. Total gradient
vol should be equal to approx 15 bed vol (see Note 2).
3. Elute with additional pH 4 buffer until the column effluent pH is
stable.
4. If the total quantity of added protein is not completely recovered,
elute with a small vol (c5 bed vol) of the 50 mM phosphate buffer
adjusted to pH 3.5.
3.2.3. Affinity Gradient Elution with Imidazole
1. Equilibrate the column first with 5 bed vol of 20 mM sodium phos-
phate (or HEPES), 0.5M NaCl, pH ‘7, containing 20 mM imidazole.
Now, equilibrate the column with 5-10 bed vol of 2 mMimidazole in
the same buffer. (See Note 3.)
2. After sample application, elute with 2.5 bed vol of 2 mMimidazole in
20 mM sodium phosphate (or HEPES), 0.5M NaCl, pH ‘7.
3. Now elute with a linear gradient to 20 mM imidazole in 20 mM
sodium phosphate (or HEPES), 0.5M NaCl, pH 7. Total imidazole
gradient vol should equal 15 bed ~01s. (See Notes 4,5.)
3.3. Evaluation of Metal Ion Exchange
or Transfkr from the Stationary Phase
to the Eluted PeptidelProtein
1. Use trace quantities of radioactive metal ions (e.g., 65Zn) to label the
stationary phase (i.e., immobilized) metal ion pool (seesection 3.1.).
After elution of adsorbed proteins (see Section 3.2.), determine the
total quantity of radioactive metal ions transferred to eluted proteins
from the stationary phase (by use of a gamma counter).
2. To avoid the use of radioactive metal ions, the transfer of metal ions
from the stationary phase to apo (metal-free) peptides present ini-
tially in the starting sample may be determined by either of two meth-
ods of soft ionization mass spectrometry. Both electrospray ionization
mass spectrometry (25) and matrix-assisted UV laser desorption time-
of-flight mass spectrometry (14,23-25) have been used to detect pep
tide-metal ion complexes (fig. 5). Both techniques are rapid (~10 min),
sensitive (pmoles), and are able to address metal-binding stoichiometry.