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Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory
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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.
Keywords (Audience):
Upper-Division UndergraduateKeywords (Domain):
Laboratory InstructionKeywords (Pedagogy):
Hands-On Learning / ManipulativesKeywords (Subject):
Biophysical ChemistryCiting Articles
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This article has been cited by 2 ACS Journal articles (2 most recent appear below).
Integrated Chemistry and Biology for First-Year College Students
Beth R. J. Abdella, Mary M. Walczak, Kim A. Kandl, and Jeffrey J. SchwinefusJournal of Chemical Education2011 Article ASAPIntegrated Chemistry and Biology for First-Year College Students
Beth R. J. Abdella, Mary M. Walczak, Kim A. Kandl, and Jeffrey J. SchwinefusJournal of Chemical Education2011 Article ASAPA three-course sequence for first-year students that integrates beginning concepts in biology and chemistry has been designed. The first two courses that emphasize chemistry and its capacity to inform biological applications are described here. The ...
The Effect of Ethylene Glycol, Glycine Betaine, and Urea on Lysozyme Thermal Stability
Jeffrey J. Schwinefus, Elizabeth J. Leslie, and Anna R. NordstromJournal of Chemical Education2010 87 (12), 1393-1395The Effect of Ethylene Glycol, Glycine Betaine, and Urea on Lysozyme Thermal Stability
Jeffrey J. Schwinefus, Elizabeth J. Leslie, and Anna R. NordstromJournal of Chemical Education2010 87 (12), 1393-1395The four-week student project described in this article is an extension of protein thermal denaturation experiments to include effects of added cosolutes ethylene glycol, glycine betaine, and urea on the unfolding of lysozyme. The transition temperatures ...
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- Received: August 03, 2009
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