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  Home > JCE Print > Journal of Chemical Education > Issues > 2008  > January  >
In the Laboratory
Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory
Jeffrey J. Schwinefus, Nathaniel J. Schaefle, Gregory W. Muth, and Gary L. Miessler
Department of Chemistry, St. Olaf College, Northfield, MN 55057

Christopher A. Clark
3M Drug Delivery Systems Division, St. Paul, MN 55144

Cover
January 2008
Vol. 85 No. 1
p. 117
Abstract
As part of an effort to infuse our physical chemistry laboratory with biologically relevant, investigative experiments, we detail four integrated thermodynamic experiments that characterize the denaturation (or unfolding) and self-interaction of hen egg white lysozyme as a function of pH and ionic strength. Students first use Protein Explorer to examine the structure of lysozyme and its charge dependence on pH. Student groups are then assigned one of four 45 mM sodium acetate buffers: pH 3.6 with either 0 or 60 mM NaCl or pH 4.6 with either 0 or 60 mM NaCl. Using their assigned buffers, student groups determine the second virial coefficient of lysozyme using laser light scattering and gel permeation chromatography. Student groups also determine the van't Hoff enthalpy of unfolding by UV absorbance thermal denaturation and compare their results to the van't Hoff enthalpy and calorimetric enthalpy of unfolding determined by differential scanning calorimetry. We stress the use of complementary techniques to determine these thermodynamic variables for student validation of experimental results. Students present their results in a peer reviewed, journal-style report and, using the data from the entire class, must determine the dependence of the lysozyme second virial coefficient and unfolding enthalpies on pH and ionic strength. Upon completion of these experiments, students are anticipated to appreciate the pH dependence of protein charge and screening of protein electrostatic repulsion with ionic strength.
Supplement
Student handouts and instructor notes containing the theory behind each experiment, the instructions for lysozyme solution preparation, use of the instruments, and data analysis are available.
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Citation
Schwinefus, Jeffrey J.; Clark, Christopher A.; Schaefle, Nathaniel J.; Muth, Gregory W.; Miessler, Gary L. J. Chem. Educ. 2008, 85, 117.
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Keywords
Biophysical Chemistry; Calorimetry / Thermochemistry; Chromatography; Hands-On Learning / Manipulatives; Inquiry-Based / Discovery Learning; Laboratory Instruction; Lasers; Physical Chemistry; Proteins / Peptides; Spectroscopy; Thermodynamics; Upper-Division Undergraduate
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History
Created:
Last Updated:
12/4/2007
12/12/2007
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