Signal and binding. II. Converting physico-chemical responses to macromolecule-ligand interactions into thermodynamic binding isotherms.
Physico-chemical titration techniques are the most commonly used methods in characterizing molecular interactions. These methods are mainly based on spectroscopic, calorimetric, hydrodynamic, etc., measurements. However, truly quantitative physico-chemical methods are absolutely based on the determination of the relationship between the measured signal and the total average degree of binding in order to obtain meaningful interaction parameters. The relationship between the observed physico-chemical signal of whatever nature and the degree of binding must be determined and not assumed, based on some ad hoc intuitive relationship/model, leading to determination of the true binding isotherm. The quantitative methods reviewed and discussed here allow an experimenter to rigorously determine the degree of binding and the free ligand concentration, i.e., they lead to the construction of the thermodynamic binding isotherm in a model-independent fashion from physico-chemical titration curves.
Biophysical Characterization of Nanoparticle-Protein Interactions by Fluorescence Quenching Titration: Limitations, Pitfalls, and Application of a Model-Free Approach for Data Analysis
Interactions of proteins with synthetic nanoparticles (NPs) can be investigated through a number of biophysical tools which are based on the principles of fluorescence. Among these, steady-state fluorescence quenching titration is one of the most popular techniques available. However, although not typically recognized, the multisite nature of NP-protein interactions prevents a straightforward application of the fluorescence quenching methodology. In this Chapter, some of the limitations and pitfalls of fluorescence quenching titration are therefore discussed and illustrated with simulations. It is demonstrated that commonly used analysis methods of fluorescence quench data are overly simplistic and often unsuitable for a true quantification of the interactions. Using both simulated and experimental data, it is shown how NP-protein association can be quantified instead through application of a rigorous model-independent method of analysis. Similar considerations may hold in the implementation of other biophysical methods to study NP-protein interactions.
Catalytic removal of tert-butyldimethylsilyl (TBS) ether by PVP-I
Abstract A mild, efficient and rapid protocol the deprotection of alcoholic TBDMS ethers using PVP-1 as catalyst in methanol, the procedure of deprotection of various TBDMS ethers were found to be very convenient, easy work-up, high yielding.