A Note on the use of Steady–State Fluorescence Quenching to Quantify Nanoparticle–Protein Interactions

Alioscka A. Sousa

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].