A Note on the use of Steady–State Fluorescence Quenching to Quantify Nanoparticle–Protein Interactions
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Steady–state fluorescence quenching is a commonly used technique to investigate the interactions between proteins and nanoparticles, providing quantitative information on binding affinity, stoichiometry and cooperativity. However, a failure to account for the limitations and pitfalls of the methodology can lead to significant errors in data analysis and interpretation. Thus, in this communication we first draw attention to a few common pitfalls in the use of fluorescence quenching to study nanoparticle–protein interactions. For example, we discuss a frequent mistake in the use of the Hill equation to determine cooperativity. We also test using both simulated and experimental data the application of a model–independent method of analysis to generate true thermodynamic nanoparticle–protein binding isotherms. This model–free approach allows for a quantitative description of the interactions independent of assumptions about the nature of the binding process [Bujalowski W, Lohman TM (1987) Biochemistry 26: 3099; Schwarz G (2000) Biophys. Chem. 86: 119].
KeywordsFluorescence quenching Spectroscopic titration Hill equation Gold nanoparticles Protein adsorption
This work was supported by the São Paulo Research Foundation (FAPESP grant # 2013/18481-5), São Paulo, Brazil, and by the National Council for Scientific and Technological Development (CNPq grant # 476784/2013-1), Brazil.
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