Award Date

5-1-2020

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry and Biochemistry

First Committee Member

Ronald Gary

Second Committee Member

Jun Yong Kang

Third Committee Member

Bryan Spangelo

Fourth Committee Member

Patricia Cruz

Number of Pages

62

Abstract

Be2+ ion is a biologically active metal that is capable of binding to proteins and has been shown to affect various cellular processes. When treated with Be2+, certain cancer cells display cytostatic effects. GSK3β is a regulatory kinase involved in the β-catenin pathway that may be involved in producing these cytostatic effects when Be2+ binds to it. In order to determine binding parameters between Be2+ and GSK3β, isothermal titration calorimetry (ITC) can be utilized. However, titrations at physiological pH cannot be carried out due to Be2+ unique speciation at neutral pH ranges. Significant precipitation occurs at pH 6 and higher when Be2+ forms BeOH2. Due to these limitations, titrations need to be carried out at a pH range where Be2+ is more soluble in order to fully understand beryllium binding characteristics. As a first step, titrations using a well characterized ligand need to be completed prior to testing with GSK3β.

Titrations between BeSO4 and EDTA were carried out at pH 5.50 using bis-tris, piperazine, acetic acid, and 2-(N-morpholino)ethanesulfonic acid (MES) buffers. However, these titrations produced unexpected apparent equilibrium constant (KITC) and binding enthalpy (ΔHITC) results that were both buffer type and buffer concentration dependent. Previous studies have concluded that certain biological buffers can form complexes with metals, such as Be2+ complexing with acetic acid buffer. It is possible to determine binding parameters (KMB and ΔHMB) between metals and biological buffers using ITC. This study aimed at determining if Be2+ was forming complexes with the biological buffers used, and if this metal-buffer complex formation was contributing to the unexpected binding parameters that were observed. To accomplish this objective, KMB and ΔHMB values were calculated.

The results indicate that Be2+ had the strongest affinity with bis-tris, followed by piperazine, then acetic acid, and negligible binding was seen with MES. Further data analysis of metal to buffer control titrations was also conducted after buffer and temperature dependent results were observed. Control titrations consisted of Be2+ to buffer titrations where EDTA was not present. These control titrations exhibited slow kinetics that appeared to be caused by Be2+ dissociation from bis-tris, piperazine, and acetic acid. Similar results were not obtained with titrations utilizing MES. Activation energies (Ea) of these observed metal-buffer dissociations of Be2+ and bis-tris, piperazine, and acetic acid were calculated with Ea ranging from 24 - 32 kcal/mol, depending on buffer type. Together, the experimental KMB, ΔHMB, and Ea results show Be2+ can form a complex with bis-tris, piperazine, and acetic acid. These data provide a clear explanation that competing equilibria from Be2+ and buffer complexation contributed to buffer-related variability in initial Be2+ and EDTA KITC and ΔHITC results. Future studies using proteins such as GSK3β and utilizing Be2+ as a titrant can now take into consideration the complex-forming capabilities of the buffers used in this study in order to minimize competing binding equilibria measured by ITC.

Keywords

Beryllium; Biological Buffers; EDTA; Isothermal Titration Calorimetry

Disciplines

Biochemistry | Biophysics | Chemistry

File Format

pdf

File Size

1.6 MB

Degree Grantor

University of Nevada, Las Vegas

Language

English

Rights

IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/

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