Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

118 Lopez and Makhatadze
The excess heat capacity defined C exc
p (T) as:
C exc
p (T) = [∆H(T) 2 / R·T 2 ]· [K / (1+K) 2 ] (7)
where the enthalpy function is defined as
∆H(T) = ∆Hfit(Tt) + ∆Cp · (T – Tt) (8)
There are seven fitted parameters: Tt, ∆Hfit, ∆Cp, AN, AU, BN, and BU. In order to analyze the data according to these equations, the reversibility of
unfolding reaction should be established experimentally by reheating the sample.
If more than 80% of the original signal is recovered the reaction can be considered
as reversible. For the analysis of the irreversible transitions, see ref. 7.
4. Notes
1. The DSC cell should be cleaned regularly. This can be accomplished in mist
cases by filling the cells with 10% sodium dodecyl sulfate (SDS) and heating it
up to 100°C, followed by a thorough rinse with distilled water. Alternatively, the
cells can be washed with 200 proof ethanol followed by a wash with distilled
water. Drying the cells is not recommended.
2. The protein concentration is a very important parameter because it is required for
the quantitative analysis according to Eqs. 1–8. The extinction coefficient can be
calculated from the number of aromatic residues and disulfide bonds in a protein
using an empirical equation (ref. 8):
ε 0.
280 1%,
nm 1cm = (5690 · NTrp + 1280 · NTyr + 120 · NSS) / Mw (9)
where Mw is the molecular mass of the protein in daltons. A simple experimental
procedure for estimating the extinction coefficient is described (9).
3. The reversibility of unfolding strongly depends on the upper temperature limit
during the first scan.
References
1. Makhatadze, G. I., Medvedkin, V. N., and Privalov, P. L. (1990) Partial molar volumes
of polypeptides and their constituent groups in aqueous solution over a broad
temperature range. Biopolymers 30, 1001–1010.
2. Makhatadze, G. I. (1998) Heat capacities of amino acids, peptides and proteins.
Biophys. Chem. 71, 133–156.
3. Makhatadze, G. I. and Privalov, P. L. (1995) Energetics of protein structure. Adv.
Prot. Chem. 47, 307–425.
4. Ibarra-Molero, B., Loladze, V. V., Makhatadze, G. I., and Sanchez-Ruiz, J. M. (1999)
Thermal vs guanidine-induced unfolding of ubiquitin. An analysis in terms of the
contributions from charge-charge interactions to protein stability. Biochemistry 38,
8138–8149.
5. Biltonen, R. L. and Freire, E. (1978) Thermodynamic characterization of conformational
states of biological macromolecules using differential scanning calorimetry.
CRC Crit. Rev. Biochem. 5, 85–124.