Joel Tellinghuisen1. 1. Department of Chemistry, Vanderbilt University,Nashville,Tennessee 37235. Electronic address: joel.tellinghuisen@vanderbilt.edu.
Abstract
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 ⇔ 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 ΔH°, 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 σq is estimated from analysis of water-water blanks. RESULTS: When the product conversion exceeds 90%, the parameter relative standard errors are proportional to σq/qtot, where the total heat qtot ≈ ΔH° [M]0V0. Specifically, σK/K×qtot/σq ≈ 25 for c=10(-3)-10, ≈ 11 c(1/3) for c=10-10(4). For c>1, n and ΔH° are more precise than K; this holds also at smaller c for the product n×ΔH° and for ΔH° when n can be held fixed. Use of as few as 10 titrant injections can outperform the customary 20-40 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.
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 ⇔ 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 ΔH°, 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 σq is estimated from analysis of water-water blanks. RESULTS: When the product conversion exceeds 90%, the parameter relative standard errors are proportional to σq/qtot, where the total heat qtot ≈ ΔH° [M]0V0. Specifically, σK/K×qtot/σq ≈ 25 for c=10(-3)-10, ≈ 11 c(1/3) for c=10-10(4). For c>1, n and ΔH° are more precise than K; this holds also at smaller c for the product n×ΔH° and for ΔH° when n can be held fixed. Use of as few as 10 titrant injections can outperform the customary 20-40 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.
Authors: Brandi M Baughman; Huanchen Wang; Yi An; Dmitri Kireev; Michael A Stashko; Henning J Jessen; Kenneth H Pearce; Stephen V Frye; Stephen B Shears Journal: PLoS One Date: 2016-10-13 Impact factor: 3.240