Rapid measurement of binding constants and heats of binding using a new titration calorimeter

A new titration calorimeter is described and results are presented for the binding of cytidine 2′-monophosphate (2′CMP) to the active site of ribonuclease A. The instrument characteristics include very high sensitivity, rapid calorimetric response, and fast thermal equilibration. Convenient software is available for instrument operation, data collection, data reduction, and deconvolution to obtain least-squares estimates of binding parameters n, δHo, ΔSo, and the binding constant K. Sample through-put for the instrument is high, and under favorable conditions binding constants as large as 108m−1 can be measured. The bovine ribonuclease A (RNase)/2′CMP system was studied over a 50-fold range of RNase concentration and at two different temperatures. The binding constants were in the 105 to 106m−1 range, depending on conditions, and heats of binding ca. −15,000 cal/mol. Repeat determinations suggested errors of only a few percent in n, ΔHo, and K values over the most favorable concentration range.