User`s manual
62 Chapter 6: Sample Experiments
Calorimetry Sciences Corp.
CSC 5300 N-ITC III 63
User’s Manual
where t is given in °C
†
.
†
Christensen, J.J., Hansen, L.D. and Izatt, R.M. (1976) Handbook of Proton Ionization Heats and Related
Thermodynamic Quantities (John Wiley and Sons, New York).
Sample Experiment
Binding of 2’-CMP to RNase A
Sample Preparation
Prepare 4 L of 15 mM acetate buffer (pH 5.5) by dissolving 5.89 g of potassium acetate,
KC
2
H
3
O
2
(FW 98.14), in 4 L of distilled water and adjusting the pH using an HCl
solution. Prepare 5 mL of RNase A (FW 13690) solution at approximately 0.07 mM by
dissolving 4.8 mg in 5 mL of the acetate buffer. The protein solution should be dialyzed
overnight in a refrigerator against the 4 L of acetate buffer using 3500 molecular weight
cut-off dialysis tubing.
The 2’-CMP (FW 323.2) solution should be prepared the following day using the
dialysis buffer. Remove a sample of buffer (~50-100 mL) and allow it to come to
room temperature. Dissolve 8.4 mg of 2’-CMP in 20 mL of buffer for an approximate
concentration of 1.3 mM.
The precise concentrations of both solutions must be determined spectrophotometrically.
The RNase A concentration should be determined using an extinction coefcient of 9800
cm
-1
M
-1
at 280 nm (Wiseman, T., Williston, S., Brandts, J.F. and Lin, L.-N. (1989) Anal.
Biochem. 179, 131). The 2’-CMP concentration must be determined at pH 7 by diluting
1 mL of sample up to 25 mL using 100 mM phosphate buffer (pH 7.0). The extinction
coefcient is then 7400 cm
-1
M
-1
at 260 nm (ibid.).
It may be preferable to prepare the above solutions at higher concentrations initially,
determine the concentrations spectrophotometrically as described, and then use the
dialysis buffer to prepare dilutions at the test concentrations of 0.07 mM for RNase A and
1.3 mM for 2’-CMP. Excess concentrated solutions and an ample amount of buffer may
be stored for later use by freezing.