Reference no: EM131128473
1.
a) What is meant by the assumption of irreversibility in the context of binding reactions?
b) Describe the differences between the "initial rate" method and "relaxation methods" to obtain rate constants.
c) The observed rate constant for the formation of a complex is larger when the experiment is performed under conditions when the reaction must be treated as reversible rather than irreversible. Explain why this is the case.
2) In a buffered solution, the concentration of free protons is constant. Any protons that are either taken up or released during a biochemical process must come from or end up on the buffer molecules. This applies to biochemical reactions as well as ligand binding. When protons are released upon ligand binding to a protein, the protons can be considered to bind to the buffer. If protons are taken up from solution upon ligand binding, they are taken from protonated buffer molecules. As a result, if binding is monitored by ITC, the heat measured must also include the heat of ionization of the buffer if it loses or picks up protons during complex formation. An example of this is described in slides 44 and 45 of lecture 19, which we did not go through in class. Look these over before doing the following problem.
The following ITC data were obtained in studying the binding of the estrogen receptor α to duplex DNA. It was found that the observed heat of the reaction depended on the buffer in which the experiment was performed.
a) Phosphate; b) Hepes; c) Tricine; d) Tris buffers
The buffers used have different enthalpies of ionization (?Hoion)
Phosphate: 1.22 kcal/mol
Hepes: 5.02 kcal/mol
Tricine: 7.64 kcal/mol
Tris: 11.35 kcal/mol
a) In the presence of phosphate buffer, is the binding of DNA to the protein endothermic or exothermic? What is your reasoning?
b) When Tris is deprotonated (ionization), is heat released or taken up from the surroundings?
c) Are protons taken up or released upon binding of the protein and DNA, or is there no change in protonation upon forming the complex. Explain.
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