Ligand-Binding Right out of the Gate in a Biochemistry Laboratory Course: Metal Binding to a Model Chromophore at Variable pH

Abstract We have developed an introductory, biochemistry laboratory module investigating the pH and metal-dependent binding of divalent metals to the model chromophore 8-hydroxyquinoline-5-sulfonic acid (HQS). Over 3 weeks, students first measure shifts in HQS absorbance versus two metals at multiple pHs, then construct and interpret binding isotherms, and finally test follow-up hypotheses with an expanded set of metals and pHs. The student learning objectives of this laboratory module are 3-fold: (1) practice essential laboratory skills (microplate analysis, pipetting, dilutions, instrument operation, nonlinear regression, error analysis, and graphic presentation), (2) reinforce introductory biochemistry material (buffers, pH, pKa’s, intermolecular forces, and equilibrium constants), and (3) introduce equilibrium ligand binding (Kd, binding isotherms, quantitative comparisons). Assessment of student results from two laboratory assignments confirms reproducible development of essential laboratory skills with improvement in pipetting, graph and table preparation, and hypothesis generation over the 3 weeks. After lab completion, students can also successfully plot and interpret binding isotherms and the relationship between Kd values and binding affinity. Depending on available time, this laboratory can be run in 1, 2, or 3 weeks, shifting from lower levels of inquiry to greater student independence. With its low time commitment, nonspecialized laboratory equipment, adaptability, and robust student learning outcomes, this module is applicable to many introductory biochemistry laboratories.