Title

Characterization of Copper (I) Binding hermodynamics to Various Small Molecules

Date of Award

2014

Document Type

Thesis

College

College of Arts and Sciences

Department

Bachelor of Science in Chemistry- Multidisciplinary

First Advisor

Nicholas E. Grossoehme

Abstract

Copper (I) is essential for several biochemical processes within enzyme active sites such as electron transfer, dioxygen binding, catalysis, and structural activity. Although the cuprous ion is the physiologically relevant oxidation state of copper, in vitro experiments have, in large, been focused on Cu2+ because it is stable under typical laboratory conditions. Furthermore, under anaerobic conditions, Cu+ participates in a disproportionation process which favors Cu2+ by a factor of approximately 1000 relative to Cu+ . As such, careful experiment design is necessary to alleviate these complications. his research aims to provide the necessary experimental foundation to directly measure the thermodynamic forces associated with cuprous ion binding energy using isothermal titration calorimetry. To avoid the potential of Cu + oxidation, all colorimetric and calorimetric experiments were conducted in a Coy Lab glove box. Additionally, acetonitrile (MeCN) was used as a stabilizing ligand to abrogate the disproportionation equilibrium of Cu + . he spectrophotometric data veriied that the expected 2:1 bicinchoninic acid (BCA)-Cu + complex was formed under these experimental conditions. he colorimetric data provided a methodical background for subsequent calorimetric experiments that determined thermodynamic parameters of Cu + binding. In Tris bufer (pH 7.5), the average binding constant was K= 2.2 ± 0.2 × 10 6 M-1 . he average enthalpy value was ΔH = -28± 0.3 kJ/mol, and the average reaction stoichiometry (n) was 2.1 ± 0.05. In bis-tris bufer (pH 7), the average enthalpy was -34± 0.4 kJ/mol, and in Hepes bufer (pH 7), the average enthalpy was -37± 0.5 kJ/mol. he diference in enthalpy values may be due to the interactions of the Cu + -BCA complex with diferent biological bufers. My future research will continue to explore this hypothesis.

Comments

Presented at the Suddath Symposium, February, 2013

Supported by an NIH-INBRE Grant from the National Center for Research Resources and the National Institute for General Medical Sciences

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