Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

116 Lopez and Makhatadze
6. Buffers should be chosen for their low ionization enthalpy (and thus low-temperature
dependence of pK a) such as glycine (pH 2.0–3.5), sodium acetate (pH
3.5–5.0), sodium cacodylate (pH 5.5–7.0), sodium phosphate (pH 6.0–7.5).
3. Methods
3.1. Instrument Preparation
1. The instrument should be turned on at least 12 h prior to the experiment and
“thermal history” established by running the baseline scans with the cells filled
with the buffer.
2. Calibration of the instrument should be done periodically (once a year) using the
procedure provided by the manufacturer (see Note 1).
3.2. Sample Preparation
1. Purified protein should be extensively dialyzed (with several changes of buffer
every 6 h or more) against corresponding buffer.
2. Prior to the experiment insoluble particle and dust should be eliminated by centrifugation
at 13,000g. Filtration of the protein solution is not recommended.
3. Measure protein concentration (see Note 2).
3.3. Data Collection
1. Thoroughly wash both cells with buffer from the last dialysis and fill them with
the buffer. It is important to avoid any air bubbles trapped in the cell. For this,
after filling up the syringe, pump out all air bubbles. Insert the needle into the
calorimetric cell (needles are precut to a specific length so that the tip of the
needle is barely above the bottom of the cell) and slowly lower the plunger until
the solution appears in the overflow reservoir. At this point, start abruptly pumping
solution in and out of the cell. This abrupt pumping will force trapped air
bubbles out from the cell.
2. Fill both cells with the buffer and run buffer/buffer scan.
3. Refill the sample cell with the protein solution and run protein/buffer scan.
4. Rescan to check the reversibility of the unfolding (see Note 3).
3.4. Data Analysis
app 1. Subtract the protein/buffer scan from the buffer/buffer scan to obtain ∆Cp (T),
the heat-capacity difference between sample and reference cells at temperature T.
app 2. Convert ∆Cp (T) into the partial heat capacity of the protein at temperature T,
exp Cp, pr (T) as:
exp Cp, pr (T) = Cp,H2O / V —
H2O · V — app pr – ∆Cp (T) / mpr
where V — pr is the partial volume of the protein, mpr is the mass of the protein in
the calorimetric cell, V —
H2O (T) is partial molar volume of aqueous buffer, and
Cp,H2O is the heat capacity of aqueous buffer. The partial volume of the protein,
V — pr can be calculated from the amino acid composition of the protein using an