Method Development of Measuring Metal/Buffer Equilibria and Enthalpy
Poster Number
52
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Faculty Mentor
Nicholas Grossoehme, Ph.D.
Abstract
An increasing interest has risen in the biochemical community to better understand the relationship between metal ions and metalloregulatory protein transcription. To understand these interactions fully, the affinity of these proteins for the metal ions must be known. However, biological buffers provide a binding competition for the proteins, making direct measurement of metal/protein affinity difficult to achieve, as proteins must have buffers present to bind effectively. Developing a method of studying these metal and buffer interactions is essential to understanding the kinetic and thermodynamic relationships between metals and proteins. Due to a fair number of experimental complications, these metal/buffer interactions are not easy to measure directly, and require indirect measurement using a metal-binding chromophore. This approach entails first binding a metal to the chromophore in the absence of buffer to enable a comparison with the binding constant when a fixed concentration of buffer is present. This was first tested using UV-spectroscopy; however, the equilibrium between the metal and chromophore could not be seen accurately using the spectrophotometer. To address this problem, the same equilibria were recorded using fluorescence spectroscopy, which allows for a significantly decreased experimental concentration. The Zn2+ – Mag-Fura-2 system was used to pilot this method, and resulted in experimental Zn/buffer affinities that match reference values. Metal-buffer enthalpies were also explored. Isothermal titration calorimetry (ITC) was used to observe the heat exchange as metal and buffer were titrated into EDTA, a high-affinity ligand. The experimental enthalpies were corrected for coupled reactions and the enthalpy of Zn-buffer interactions were determined. While the enthalpies we observed support the validity of the method, there were certain discrepancies in the results of the reaction that warrant further study to validate the method.
Start Date
21-4-2017 2:15 PM
Method Development of Measuring Metal/Buffer Equilibria and Enthalpy
Richardson Ballroom
An increasing interest has risen in the biochemical community to better understand the relationship between metal ions and metalloregulatory protein transcription. To understand these interactions fully, the affinity of these proteins for the metal ions must be known. However, biological buffers provide a binding competition for the proteins, making direct measurement of metal/protein affinity difficult to achieve, as proteins must have buffers present to bind effectively. Developing a method of studying these metal and buffer interactions is essential to understanding the kinetic and thermodynamic relationships between metals and proteins. Due to a fair number of experimental complications, these metal/buffer interactions are not easy to measure directly, and require indirect measurement using a metal-binding chromophore. This approach entails first binding a metal to the chromophore in the absence of buffer to enable a comparison with the binding constant when a fixed concentration of buffer is present. This was first tested using UV-spectroscopy; however, the equilibrium between the metal and chromophore could not be seen accurately using the spectrophotometer. To address this problem, the same equilibria were recorded using fluorescence spectroscopy, which allows for a significantly decreased experimental concentration. The Zn2+ – Mag-Fura-2 system was used to pilot this method, and resulted in experimental Zn/buffer affinities that match reference values. Metal-buffer enthalpies were also explored. Isothermal titration calorimetry (ITC) was used to observe the heat exchange as metal and buffer were titrated into EDTA, a high-affinity ligand. The experimental enthalpies were corrected for coupled reactions and the enthalpy of Zn-buffer interactions were determined. While the enthalpies we observed support the validity of the method, there were certain discrepancies in the results of the reaction that warrant further study to validate the method.
