Microplate fluorescence quenching for high throughput screening of affinity constants – Serum albumins and zearalenones case study

Measurements of changes in fluorescence signal is one of the most commonly applied methods for studying protein-ligand affinities. These measurements are generally carried out using cuvettes in spectrofluorometers, which can only measure one sample at a time. This makes screening procedures for multiple ligands and proteins extremely laborious, as each protein must be measured with multiple ligand concentrations, and usually in triplicate. Moreover, multiple equations exist to extract the affinity constants and other information from the data, and their underlying assumptions are often disregarded. In this study, the affinities of human, bovine and rat serum albumins for the mycotoxin zearalenone and five of its common derivatives were measured in 96-well microplates, allowing quick measurements of multiple samples using less reagent amounts. In comparison to measurements using a cuvette in a spectrofluorometer, the microplate method was shown to reproduce the affinity constants accurately. The results were discussed in terms of common pitfalls regarding experimental setup and available equations to analyze protein-ligand binding in fluorescence quenching assays. The commonly used Stern-Volmer equation was discussed in detail and the results used to show how inaccurate it is when a fluorescent protein-ligand complex is formed, and when other underlying approximations are ignored.