title: A Note on the use of Steady-State Fluorescence Quenching to Quantify Nanoparticle-Protein Interactions.
abstract: 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].
authors.affiliation: Department of Biochemistry, Federal University of São Paulo, São Paulo, SP, 04044, Brazil. alioscka.bioq@gmail.com.
authors.forename: Alioscka A
authors.initials: AA
authors.lastname: Sousa
mesh_terms: D002918:Chymotrypsin / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D006046:Gold; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
keywords: Fluorescence quenching; Gold nanoparticles; Hill equation; Protein adsorption; Spectroscopic titration