Abstract
A Microsoft Excel workbook has been developed to simplify the quantitative analysis of experimentally measured titration curves for diprotic and triprotic amino acids such as glycine, arginine, histidine, and glutamate. Students perform the titration, enter the data into the worksheet, and manually adjust the resulting pKa values to achieve the closest agreement between the experimental and calculated titration curves. At the same time, the students can observe changes in the speciation diagram that illustrates the transitions of differently protonated molecular species during the titration. Through a combination of graphical visualization and analysis, supported by computer software, students learn the most important aspects of the protonation equilibria of amino acid solutions. The knowledge gained can serve as the basis for understanding the behavior of more complex polyprotic acids in aqueous solution and the pKa values of amino acid residues in proteins.
Highlights
Proton transfer in aqueous systems plays a fundamental role in many biochemical processes, such as enzyme catalysis,[1−3] conformational changes of macromolecules,[4−6] maintenance of pH gradients in cell compartments,[7] formation of some noncanonical DNA structures,[8] and others
The first chart appears under the sheet “Chart” and can be copied using the “Copy graph” button, while the second can be found in sheets “Species1” and “Species2”
Since the transfer of protons presents a part of complicated chemical and biochemical processes in aqueous solutions, laboratory titration experiments with single amino acids are the best starting point for introducing this subject to students in a biochemistry course
Summary
Proton transfer in aqueous systems plays a fundamental role in many biochemical processes, such as enzyme catalysis,[1−3] conformational changes of macromolecules,[4−6] maintenance of pH gradients in cell compartments,[7] formation of some noncanonical DNA structures,[8] and others. In contrast to monoprotic acids, it is not a trivial task to determine pKa values of polyprotic amino acids quantitatively For this reason, students are commonly encouraged to analyze titration curves semiquantitatively using a graphic method where they become familiar with basic features of titration curves such as buffering regions and isoelectric points. A second, more advanced step is an exact quantitative analysis of the whole titration curve which would strengthen students’ understanding of protonation equilibria. Such a quantitative analysis relates a mathematical description and the use of computer software to the experimental data, and it may significantly improve the understanding of these topics. Graphical simulations of acid−base pH titrations, using a computer spreadsheet, and applications for educational purposes are available.[25−30] A theoretical description of titration curves, using a direct single closed-form expression without assumptions and segmentations (dissection of titration curve into two or more segments), is usually problematic since it involves solving a high-order polynomial.[27,31] Less complex approaches for data fitting have been proposed that are based on indirect methods using simplified assumptions or functions without direct physical significance.[32−35] to our knowledge, there are presently no simple tools for students that would combine graphical simulation, theoretical description, and data fitting for amino acid titrations
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