A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.

Fast and Robust Quantification of Detergent Micellization Thermodynamics from Isothermal Titration Calorimetry.

Detergents are widely used in modern in vitro biochemistry and biophysics, in particular to aid the characterization of integral membrane proteins. An important characteristic of these chemicals in aqueous solutions is the concentration above which their molecular monomers self-associate to form micelles, termed the critical micellar concentration (CMC). Micelles are supramolecular assemblies arranged with the hydrophobic portions oriented inward and the hydrophilic head groups positioned outward to interact with the aqueous solvent. Knowledge of the CMC is not only of practical relevance but also of theoretical interest because it provides thermodynamic insights. Isothermal titration calorimetry (ITC) is a powerful method to determine CMCs, as it furnishes additional information on the enthalpy and entropy of micellization. Here we describe our extension of previous methods to determine CMCs and other thermodynamic parameters from ITC demicellization curves. The new algorithm, incorporated into the stand-alone software package D/STAIN, analyzes ITC demicellization curves by taking advantage of state-of-the-art thermogram-integration techniques and automatically providing rigorous confidence intervals on the refined parameters. As a demonstration of the software's capabilities, we undertook ITC experiments to determine the respective CMCs of n-octyl ß-d-glucopyranoside (OG), n-dodecyl ß-d-maltopyranoside (DDM), and lauryldimethylamine N-oxide (LDAO). Motivated by the fact that in vitro membrane protein studies often require additives such as precipitants (e.g., polyethylene glycol (PEG)), we also carried out ITC demicellization studies in the presence of PEG3350, finding in all cases that PEG had significant effects on the thermodynamics of detergent micellization.

Model-Free Analysis of Critical Micellar Concentrations for Detecting Demixing in Surfactant Mixtures.

Aqueous mixtures of two or more surfactants are often employed for research or industrial purposes because such mixtures offer advantages over single-surfactant systems. This is particularly true for mixtures of fluorocarbon (FC) and hydrocarbon (HC) surfactants, which display a broad range of mutual miscibilities in mixed micelles. Unfortunately, the prediction and even the experimental elucidation of the micellar mixing behavior of surfactant mixtures remain challenging, as evidenced by conflicting results and conclusions derived from diverse, and often complex, mixing models. One of the most intriguing questions is whether certain combinations of FC and HC surfactants form only one type of mixed micelle or rather demix into two micelle populations, namely, FC-rich and HC-rich ones. Here, we demonstrate a novel approach to the model-free analysis of critical micellar concentrations (CMCs) of surfactant mixtures that is based on a fit of the experimental data with cubic splines using a stringent thermodynamic criterion for mixing. As a proof of principle, we analyze CMC values determined by isothermal titration calorimetry and confirm the conclusions with the aid of combined 1H- and 19F-NMR spectroscopy. Specifically, we show that aqueous mixtures of an FC maltoside and an HC maltoside conform with the assumption of only one type of micelle regardless of the mixing ratio, whereas combining the same FC surfactant with an HC surfactant carrying a zwitterionic phosphocholine headgroup gives rise to two coexisting micelle populations at high mole fractions of the FC maltoside.