Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

80 Weljie and Vogel
VIS spectrophotometer and acquiring an absorption spectrum between 250 and
320 nm. It may also be worthwhile to acquire a UV/VIS spectrum of the buffer
between 300 and 450 nm to ensure that there is no transfer of tryptophan-fluorescence
emission to the buffer. If one is examining the interaction of charged species,
the addition of 50–100 mM KCl or NaCl may be beneficial to prevent
nonspecific interactions. Finally, phosphate buffers are to be avoided in studies
involving Ca 2+ because of the precipitation of calcium phosphate solid.
4. For quenching studies, a saturated stock solution of the chosen quenching agent
is required. For example, a stock solution of potassium iodide can be made by
adding 30.0 g of KI to 21.0 mL of water, to form 30.0 mL of a 6.0 M solution (see
Note 2).
3. Method
3.1. Sample Preparation
1. Purified, dry protein is weighed and then dissolved in the chosen buffer. Care
must be taken to ensure that the pH does not vary significantly. This is especially
necessary for calcium-binding proteins which often have significant numbers of
acidic residues in the calcium binding regions (see Note 3).
2. The concentration of the sample must be quantitatively determined, as the dry
mass is not a reliable indicator of protein mass. The method of choice for such
concentration determinations of proteins (or peptides) without tryptophan and
tyrosine is quantitative amino acid analysis. If the protein sequence is known, the
absorbance at 280 nm can be used if the protein contains tryptophan or tyrosine
residues. The extinction coefficient can be calculated as:
ε280 = x εtyr + y εtrp + z εcys where x, y, and z are the numbers of each residue in the sequence, and the value of
εtyr is 1280 M –1cm –1 , εtrp is 5690 M –1cm –1 , and εcys (cystine) is 120 M –1cm –1 . (13)
(see Note 4).
3. If a number of samples are to be made of the same protein or peptide, a 100X
stock solution can be made, and then a sample pool made with a volume slightly
greater than the total volume of all samples. For example, if 10 3.0 mL samples of
protein are needed, then a 30.5-mL pool might be made, and each sample made
from this pool. This will help ensure that the concentration of each sample
remains consistent, which is especially important when comparing spectra of
the same species under different conditions. Ensure that the volume used also
takes into account other reagents that might be added, such as calcium stock
solutions (see Note 5 for suggested stock concentrations).
4. Samples involving binding (calcium and or protein/peptide ligands) should be
allowed to come to a complete equilibrium prior to spectrum acquisition. For
stable species, samples can be prepared the day prior to use, and then stored at 4°
overnight. Ensure that the samples are allowed to equilibrate to room tempera-