Optimizing isothermal titration calorimetry protocols for the study of 1:1 binding: Keeping it simple

Background: Successful ITC experiments require conversion of cell reagent (titrand M) to product and production or consumption of heat. These conditions are quantified for 1:1 binding, M + X double left right arrow MX. Methods: Nonlinear least squares is used in error-propagation mode to predict the precisions with which the key quantities - binding constant K, reaction enthalpy Delta H degrees, and stoichiometry number n - can be estimated over a wide range of the dimensionless quantity that governs isotherm shape, c = K[M](0). The measurement precision sigma(q) is estimated from analysis of water-water blanks. Results: When the product conversion exceeds 90%, the parameter relative standard errors are proportional to sigma(q)/q(tot), where the total heat q(tot) approximate to Delta H degrees [M](0) V-0. Specifically, sigma(K)/Kxq(tot)/sigma(q)approximate to 25 for c = 10(-3) - 10, approximate to 11 c(1/3) for c = 10-10(4). For c > 1, n and Delta H degrees are more precise than K; this holds also at smaller c for the product n x Delta H degrees and for Delta H degrees when n can be held fixed. Use of as few as 10 titrant injections can outperform the customary 2040 while also improving productivity. Conclusion: These principles are illustrated in experiment design using the program ITC-PLANNER15. General significance: Simple quantitative guidelines replace the "c rules" that have dominated the literature for decades. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences - Principles and Applications, edited by Fadi Bou-Abdallah. (C) 2015 Elsevier B.V. All rights reserved.