Effect of pH and Metal Ions on Protein-Metal Complexation Modeled by Fluorescence Quenching

Obtaining and analyzing fluorescence spectra is a valuable skill for students taking a course in enviro-analytical chemistry. This experiment allowed students to gain an understanding of metal complexation at varying pH using a fluorescence quenching technique. Students collected fluorescence spectra of BSA quenched by a range of Cu or Pb concentrations at two pH levels and conducted a Stern-Volmer analysis. Increasing the quenching agent concentration caused a decrease in the fluorescence intensity. Students recorded and plotted the fluorescence intensities at the peak maximum (F max) against quenching agent concentration. The Stern-Volmer equation and modified Stern-Volmer equation were used to obtain complexation parameters: the Stern-Volmer constant of association (Ksv), the binding constant (Ka), and the stoichiometric coefficient of the metal (n). Copper demonstrated stronger binding with BSA at pH 5.1, while lead was a more effective quenching agent at pH 3.4. The degree of quenching decreased for both metals at pH 3.4, markedly more for Cu. This may be due to enhanced aggregation or unfolding of BSA at a lower pH, altering accessibility to binding sites. It may also be due to competitive binding with protons. Students were asked several questions related to their findings and to seek out additional research to support their ideas. Overall, students gained a molecular-level understanding of BSA-metal binding and the implications of protein conformation and denaturation at different pH levels. The questions were also used to prompt learning about environmental implications, including impacts of binding strength on the bioavailability of metal ions in the human body and aquatic systems.