Binding curves by continuous gradient flow-mix calorimetry

Abstract

Steady-state flow-mix calorimetric methods require large amounts of sample and time to obtain data at variable reactant ratios. The use of continuous concentration gradients allows rapid generation of data with chemicals consumption comparable to other calorimetric techniques. An experimental set-up able to generate a linear concentration gradient of one reactant, while keeping the other concentration constant during the experiment, permits a complete titration in a single experiment. Since the calorimeter is not operating under steady-state conditions, the output is affected by the dynamics of the instrument. Therefore, it is necessary to correct the experimental curve by deconvolution to obtain the true signal. Titration of the carboxyl group of glycine with hydrochloric acid, as well as the binding of ribonuclease A to two different nucleotides prove the efficacy of this new methodology.