Isothermal Titration Calorimetric Investigations of Biochemical Systems
We continue to develop a number of experiments using the isothermal titration microcalorimeter (ITC) obtained from MicroCal, Inc., Northampton, Massachusetts for the chemisty and biochemistry curriculum. The purchase of the equipment was supported by The Camille and Henry Dreyfus Foundation through its Special Grant Program in the Chemical Sciences and the National Science Foundation through an Instrumentation and Laboratory Improvement Grant in the Division of Undergraduate Education.
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A Calorimetric Study of the Ribonuclease System
This investigation involves the interaction of the enzyme ribonuclease (RNase) with the substrate analogue cytidine monophosphate (CMP). The binding of native RNase, RNase S (created by limited proteolysis of RNase with subtilisin), the S-peptide and the S-protein with CMP can be studied with the ITC to yield a complete thermodynamic profile. This profile includes, by direct experimental determination: delta H, the enthalpy change; K, the equilibruim binding constant; n, the number of binding sites; and by calculation: delta S, the entropy change; and delta G, the free energy change. The binding of the S-peptide to the S-protein also has been investigated and a complete thermodynamic profile obtained.
Calorimetric Studies of the Binding of Metals to Tetracycline
Most recently we have been studying the effect of the binding of metal ions to tetracycline based on the work of Ohyama and Cowan, Inorganic Chemistry 34, 3083-3086 (1995). The binding of Ca(II), Mg(II), Sr(II), and Mn(II) has yielded interesting thermodynamic data which illustrates the importance of water structure on the binding phenomenon.
Elliott was a Chemistry major at Williams College. His hometown is Olympia, Washington.
Abstract of the paper presented at the Satellite Meeting "2001: Biochemistry Education for the Millennium" held in conjuction with the 17th International Congress of Biochemistry and Molecular Biology, San Francisco, August 1997.
THE ISOTHERMAL TITRATION MICROCALORIMETER IN THE BIOCHEMISTRY AND MOLECULAR BIOLOGY LABORATORY Lawrence J. Kaplan, Dale Woodnutt, Elliott H. Sohn, and Graham Dresden, Department of Chemistry, Williams College, Williamstown, MA, 01267
A fast, reliable isothermal titration microcalorimeter (the MCS-ITC from MicroCal, Inc.) which measures heat directly, makes possible the rapid determination in a single experiment of a complete thermodynamic profile (the equilibrium constant, K, the enthalpy change, DH, the free energy change, DG, the entropy change, DS, and the number of binding sites, n) of a wide variety of systems with high precision and accuracy. Since the "heat signal" is an almost universal property of binding reactions, ITC can be used to monitor the binding of ligands to (macro)molecules as well as macromolecular interactions. A variety of experiments have been developed which may be incorporated into the laboratory program of biochemistry and molecular biology courses. These include: the binding of the inhibitor cytidine monophosphate to native ribonuclease A (RNaseA), RNase S, S-protein, and S-peptide (formed by digestion with subtilisin); the relative enthalpic and entropic contributions of the binding of metal ions, Mg(II), Mn(II), Ca(II), and Sr(II), to tetracycline to assess the differing modes of binding and changes in solvation upon complexation; and the binding of specific carbohydrates by the lectin Con A allowing the determination of the nature of the binding pocket. (Partial support: The Camille and Henry Dreyfus Foundation Special Grant Program in the Chemical Sciences)
Abstract of the paper presented at the 213th National Meeting of the American Chemical Society, San Francisco, April 1997.
THE USE OF THE ISOTHERMAL TITRATION MICROCALORIMETER IN THE UNDERGRADUATE CHEMISTRY AND BIOCHEMISTRY LABORATORY. Lawrence J. Kaplan, Dale Woodnutt and Elliott H. Sohn, Department of Chemistry, Williams College, Williamstown, MA 0126
The recent development of a fast, reliable isothermal titration microcalorimeter (the MCS-ITC from MicroCal, Inc.) which measures heat directly, allows the rapid determination, in a single experiment, of a complete thermodynamic profile (the equilibrium constant, K, the enthalpy change, deltaH, the free energy change, deltaG, the entropy change, deltaS, and the number of binding sites, n) of a wide variety of systems with high precision and accuracy. Since the "heat signal" is an almost universal property of binding reactions, ITC can be used to monitor the binding of ligands to (macro)molecules as well as macromolecular interactions. Experiments such as the following may be incorporated into the laboratory program in physical, inorganic, or biochemistry courses: the binding of the inhibitor cytidine monophosphate to native ribonuclease A, RNase S, S-protein, and S-peptide (formed by digestion with subtilisin); the relative enthalpic and entropic contributions of the binding of metal ions, Mg(II), Mn(II), Ca(II), and Sr(II), to tetracycline to assess the differing modes of binding and changes in solvation upon complexation; and the binding of specific carbohydrates by the lectin Con A allowing the determination of the nature of the binding pocket.
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