An Experiment Illustrating the Change in Ligand pK(a) upon Protein Binding

作者：Chenprakhon Pirom; Panijpan Bhinyo; Chaiyen Pimchai^*

来源：Journal of Chemical Education, 2012, 89(6): 791-795.

DOI：10.1021/ed2006482

• Graduate Education/Research
• Upper-Division Undergraduate
• Analytical Chemistry
• Biochemistry
• Laboratory Instruction
• Acids/Bases
• Bioanalytical Chemistry
• Biophysical Chemistry
• Proteins/Peptides
• Thermodynamics

The modulation of ligand plc due to its surrounding environment is a crucial feature that controls many biological phenomena. For example, the shift in the pK(a) of substrates or catalytic residues at enzyme active sites upon substrate binding often triggers and controls enzymatic reactions. In this work, we developed an experiment using spectrophotometric method to demonstrate how ligand pK(a) values can be influenced by specific interactions in the protein-binding pocket using riboflavin binding protein (RP) and its ligands (riboflavin, RF, and neutral red, NR). A direct plot of observed absorbance versus pH was analyzed by nonlinear regression. The pK(a) values of free and RP-bound RF were determined to be 10.0 +/- 0.1 and similar to 13.3 respectively, and the pK(a) values of free and RP-bound NR were 6.8 +/- 0.1 and 7.8 +/- 0.1, respectively. This laboratory clearly demonstrates that the environment of a protein-binding site can affect the pK(a) value of a ligand. The experiment can be adapted or used as-is for undergraduate students in biochemistry or chemistry (analytical students in biochemistry and related fields. or physical chemistry) or first-year graduate

• 出版日期2012-6